Method of making a formulation for deactivating nucleic acids

ABSTRACT

The disclosure relates to formulations for use in deactivating nucleic acids and methods of making and using the same.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/531,924, filed on Jun. 25, 2012, now pending, which is a divisionalof U.S. patent application Ser. No. 11/073,085, now pending, filed onMar. 4, 2005, which claims the benefit of U.S. Provisional ApplicationNo. 60/550,749, filed on Mar. 5, 2004, the contents of each of whichapplications are hereby incorporated herein by reference in theirentirety.

FIELD

The present disclosure relates to formulations, methods and kitscontaining or employing an agent for use in deactivating nucleic acidspresent on a surface or in a solution.

BACKGROUND

Procedures for qualitatively or quantitatively determining the presenceof particular organisms or viruses in a test sample routinely rely uponnucleic acid-based probe testing. To increase the sensitivity of theseprocedures, an amplification step is often included to increase the copynumber of potential nucleic acid target sequences present in the testsample. During amplification, polynucleotide chains containing thetarget sequence and/or its complement are synthesized in atemplate-dependent manner from ribonucleoside or deoxynucleosidetriphosphates using nucleotidyltransferases known as polymerases. Thereare many amplification procedures in general use today, including thepolymerase chain reaction (PCR), Q-beta replicase, self-sustainedsequence replication (3SR), transcription-mediated amplification (TMA),nucleic acid sequence-based amplification (NASBA), ligase chain reaction(LCR), strand displacement amplification (SDA) and loop-mediatedisothermal amplification (LAMP), each of which is well known in the art.See, e.g., Mullis, “Process for Amplifying Nucleic Acid Sequences,” U.S.Pat. No. 4,683,202; Erlich et al., “Kits for Amplifying and DetectingNucleic Acid Sequences,” U.S. Pat. No. 6,197,563; Walker et al., NucleicAcids Res., 20:1691-1696 (1992); Fahy et al., “Self-sustained SequenceReplication (3SR): An Isothermal Transcription-Based AmplificationSystem Alternative to PCR,” PCR Methods and Applications, 1:25-33(1991); Kacian et al., “Nucleic Acid Sequence Amplification Methods,”U.S. Pat. No. 5,399,491; Davey et al., “Nucleic Acid AmplificationProcess,” U.S. Pat. No. 5,554,517; Birkenmeyer et al., “Amplification ofTarget Nucleic Acids Using Gap Filling Ligase Chain Reaction,” U.S. Pat.No. 5,427,930; Marshall et al., “Amplification of RNA Sequences Usingthe Ligase Chain Reaction,” U.S. Pat. No. 5,686,272; Walker, “StrandDisplacement Amplification,” U.S. Pat. No. 5,712,124; Notomi et al.,“Process for Synthesizing Nucleic Acid,” U.S. Pat. No. 6,410,278;Dattagupta et al., “Isothermal Strand Displacement Amplification,” U.S.Pat. No. 6,214,587; and Lee et al., Nucleic Acid AmplificationTechnologies: Application To Disease Diagnosis (1997).

Nucleic acid products formed during an amplification procedure (i.e.,amplicon) can be analyzed either during the course of the amplificationreaction (real-time) or once the amplification reaction has beengenerally completed (end-point) using detectable probes. While theprobes are designed to screen for target-containing amplicon, otherproducts may be produced during an amplification procedure (e.g.,primer-dimers formed in a typical PCR reaction) that have the potentialto interfere with the desired amplification reaction. Followingcompletion of the amplification procedure and exposure to detectableprobes, the resulting reaction mixture is discarded.

During the steps of an assay or synthesis procedure which includes anamplification procedure, it is possible to contaminate work surfaces orlaboratory equipment with nucleic acids used or formed in the assaythrough spills, mishandling, aerosol formation, etc. This nucleic acidcan then carry-over and contaminate future amplification and othernucleic acid assay procedures performed using the same laboratoryequipment and/or on the same work surfaces. The presence of carryoverproducts can result in the unwanted consumption of amplificationreagents or, in the case of target-containing amplicon from a previousamplification procedure, it can lead to an erroneous result, asamplification procedures are capable of detecting the presence of evenminute amounts of target nucleic acid. In the case of a syntheticamplification reaction, the desired nucleic acid product may becomecontaminated by carry-over products and/or synthesis yields may bereduced.

Various methods have been devised to limit carryover contamination. APCR amplification product, for example, can be deactivated from furtheramplification by irradiation with UV light. See Ou et al.,BioTechniques, 10:442-446 (1991); and Cimino et al., Nucleic Acids Res.,19:99-107 (1991). Such irradiation in the absence or presence of a DNAbinding photoactivatable ligand (e.g., isopsoralen) makes the productDNA nonamplifiable but retains the specific hybridization property. Inaddition, use of a 3′-ribose primer in a PCR reaction produces nucleicacid that can be readily destroyed by an alkali (e.g., NaOH). See Walderet al., Nucleic Acids Res., 21:4339-4343 (1993). Similarly, otherprocedures are used to produce specific modified nucleic acids that canbe selectively destroyed by treatment with a specific enzyme. Suchmodified nucleic acids have been produced by amplification in thepresence of dUTP as a substrate in a PCR reaction. Deoxy U-containingproduct DNA can be deactivated by a U-specific enzyme making the DNAnonamplifiable. See Integrated DNA Technologies Technical Bulletin,Triple C primers (1992); and Longo et al., Gene, 93:125-128 (1990). Manyof these methods function well with DNA but require expensive reagentsand affect the course of the amplification procedure (e.g., requiringlonger times and specific reagents).

In a preferred method, work surfaces and laboratory equipment exposed tonucleic acid products are treated with a 50% bleach solution (i.e., ableach solution containing about 2.5% to about 3.25% (w/v) sodiumhypochlorite) to deactivate nucleic acids. See GEN-PROBE® Aptima Combo2® Assay Package Insert, IN0037 Rev. A/2003-08. While this bleachsolution is effective at deactivating nucleic acids present on treatedsurfaces, it tends to create noxious fumes in poorly ventilated areasand corrodes laboratory equipment over time. Therefore, it is an objectof the present disclosure to provide a formulation containing a nucleicacid deactivating agent that is stable in solution, has a tolerableodor, and which is non-corrosive or is substantially less corrosive thana standard 50% bleach solution.

SUMMARY

The present disclosure satisfies this objective by providing aformulation that contains or can be combined with a nucleic aciddeactivating agent (“deactivating agent”) in an amount sufficient todeactivate nucleic acids contacted with the formulation in solution oron a solid surface. By “deactivate” is meant that the nucleic acid isaltered such that it can no longer function as it did prior todeactivation. For example, the nucleic acid may no longer be capable ofacting as a template in, or otherwise interfering with (e.g., throughthe formation of primer-dimers), an amplification reaction, binding toanother nucleic acid or protein, or serving as a substrate for anenzyme. The term “deactivate” does not imply any particular mechanism bywhich the deactivating agent of the formulation alters nucleic acids.The components of the formulation include a corrosion-inhibiting agent,a wetting agent, a solubilizing agent and, optionally, a deactivatingagent. When the formulation is comprised of all four components, thecorrosion-inhibiting agent is present in an amount sufficient to reducethe corrosiveness of the deactivating agent, the wetting agent ispresent in an amount sufficient to improve the dispersion properties ofthe deactivating agent and/or to increase the solubility of thedeactivating agent and/or other material present on a solid surface orin a solution, the solubilizing agent is present in an amount sufficientto increase the solubility of the deactivating agent, or the corrosioninhibiting agent, or the wetting agent, or various combinations thereof,and the deactivating agent is present in an amount sufficient tosubstantially deactivate nucleic acids contacted with the formulation.If the formulation does not include the deactivating agent, then theamounts of the corrosion-inhibiting agent, the wetting agent and thesolubilizing agent are concentrated to account for their decreasedconcentrations when combined with the deactivating agent and anydiluents (e.g., water) which may be used to form a final workingsolution capable of deactivating nucleic acids.

Deactivating agents of the present disclosure are selected for theirability to substantially deactivate nucleic acids present on a surfaceor in a solution, thereby preventing the nucleic acids from acting asunintended templates in an amplification reaction or otherwisecontaminating a workspace, laboratory equipment or materials, or workingsolutions. For certain applications, the deactivating agents of thepresent disclosure may be used without the corrosion-inhibiting agent,the wetting agent and/or the solubilizing agent referred to above.Preferred deactivating agents include bleach, sodium hypochlorite(NaOCl) (or hypochlorous acid (HOCl), which results when chlorine ionsare combined with water), sodium hypochlorite and sodium bromide (NaBr),dichloroisocyanurate (DCC), hydrogen peroxide (H₂O₂) and metal ions,preferably copper ions (Cu⁺⁺) (e.g., cupric sulfate (CuSO₄) or cupricacetate (Cu(CH₃COO)₂.H₂O)), hydrogen peroxide in combination with metalions and piperazine or piperazine-containing formulations, acetate orascorbate, percarbonate (2Na₂CO₃.3H₂O₂), peroxymonosulfate (KHSO₅),peroxymonosulfate and potassium bromide (KBr), hypobromite ions (OBr-)(e.g., hypobromous acid (HOBr)) and halohydantoins (e.g.,1,3-dihalo-5,5-dimethylhydantoins). Hypochlorite and hypobromite ionsmay be delivered to a solution using a salt, such as sodium.Particularly preferred are deactivating agents containing chloroniumions (Cl⁺), such as sodium hypochlorite, a component of householdbleach, or DCC. The DCC may be substantially pure or it may be part of aDCC-containing solution, such as ACT 340 PLUS 2000® disinfectant,containing sodium dichloroisocyanurate dihydrate at 40% p/p. Anadvantage of DCC is that it is less corrosive and, in some cases, moreresistant to inactivation by contaminating organic material thanhypochlorite.

While the deactivating agents of the present disclosure may be provided,alone or as part of a formulation, in any amount sufficient todeactivate nucleic acids, preferred concentration ranges ofabove-described deactivating agents are as follows: (i) from about 0.06%to about 3% (w/v), about 0.18% to about 1.8% (w/v), about 0.6% to about1.5% (w/v), or about 0.6% to about 1.2% (w/v) sodium hypochlorite, orsodium hyphochlorite and sodium bromide, where the sodiumhypochlorite:sodium bromide ratio is about 5:1 to about 1:5, about 2:1to about 1:2, or about 1:1; (ii) from about 5 mM to about 400 mM, about10 mM to about 200 mM, about 20 mM to about 100 mM, or about 40 mM toabout 80 mM DCC; (iii) from about 100 mM to about 880 mM, about 200 mMto about 880 mM, or about 250 mM to about 800 mM percarbonate; (iv) fromabout 50 mM to about 300 mM or about 100 mM to about 200 mMperoxymonosulfate or peroxymonosulfate and potassium bromide, where theperoxymonosulfate:potassium bromide ratio is about 2:1 to about 1:2 orabout 1:1. These ranges reflect concentrations in final workingsolutions to be used directly on a surface or in a solution and may beadjusted where the formulation is a concentrate. The preferredconcentration ranges of hydrogen peroxide containing formulations aredescribed below.

When chlorine is a component of the deactivating agent (e.g., sodiumhypochlorite or DCC), the potential organic load on a surface or in asolution that will be exposed to the deactivating agent is a factor indetermining the concentration of the chlorine-containing component. Thisis because organic materials, especially compounds containing primaryamine and sulfhydryl groups, react with chloronium ions and effectivelyscavenge them from solution. Therefore, when selecting the concentrationof the chlorine containing component to use in the formulation fordeactivating nucleic acids, consideration must be given not only to theexpected amount of nucleic acid on the surface or in the solution to betreated, but also to the expected organic load, as well as sources ofinterfering substances of a non-organic origin. Interfering substancesmay also affect non-chlorine based deactivating agents and, for thisreason, their influence on a deactivating agent should be evaluated whendetermining the concentration of the deactivating agent needed todeactivate nucleic acids on a surface or in a solution.

The corrosion-inhibiting agents of the formulation are selected tocounter the corrosive effects of the deactivating agent. As an example,bleach is a highly corrosive material that can damage laboratoryequipment and fixtures over time, requiring early replacement. Weunexpectedly discovered that the corrosion-inhibiting agents do notinterfere with the activity of the deactivating agents.Corrosion-inhibiting agents of the present disclosure include phosphate,borate, sodium bicarbonate, detergents and other corrosion-inhibitingagents known in the art. Particularly preferred is sodium bicarbonate.The concentration of the corrosion-inhibiting agent present in theformulation, when combined with the deactivating agent in a finalworking solution for direct use on a surface or in a solution, ispreferably in the range of from about 10 mM to about 750 mM. The pH ofthe corrosion-inhibiting agent should be selected to limit any loss inthe activity of the deactivating agent over time, yet still be effectivein reducing the corrosiveness of the deactivating agent. By way ofexample, sodium salts of phosphate were found to destabilize sodiumhypochlorite at pH 6.4 and 7.5 but not at pH 9.1 and 9.5. Conversely,sodium salts of phosphate were found to destabilize DCC at pH 9.1 and9.5 but not at pH 6.4 and 7.5.

The wetting agent is included in the formulation to ensure that thedeactivating agent makes sufficient contact with the surface beingtreated and/or to improve the solubility of the deactivating agentand/or other material that may be present on a surface or in a solutionto be decontaminated (e.g., nucleic acids, organic substances, oils orfilms, etc.). Detergents and surfactants are preferred wetting agentsbecause they reduce surface tension and allow for more complete wettingof surfaces with the deactivating agent. Additionally, detergents andsurfactants help to solubilize materials to be removed from surfaces ordeactivated in a solution. But because detergents and surfactants tendto foam, detergent and surfactant types and concentrations should beselected to limit foaming while providing good wetting andsolubilization qualities in the final working solution. Preferreddetergents and surfactants include sodium dodecyl sulfate (SDS), lithiumlauryl sulfate (LLS), Photo-Flo® 200 Solution (Eastman Kodak Company,Rochester, N.Y.; Cat. No. 146-4502), saponin, cetyl trimethylammoniumbromide (CTAB), Alconox® detergent containing 10-30% (w/w) sodiumdodecylbenzenesulfonate, 7-13% (w/w) sodium carbonate, 10-30% (w/w)tetrasodium pyrophosphate and 10-13% (w/w) sodium phosphate (Alconox,Inc., White Plains, N.Y.; Cat. No. 1104-1), MICRO-90® cleaning solutioncontaining less than 20% (w/w) glycine,N,N′-1,2-ethanediylbis-(N-(carboxymethyl)-,tetra-sodium salt, less than20% (w/w) benzenesulfonic acid, dimethyl-, ammonium salt, less than 20%(w/w) benzenesulfonic acid, dodecyl-, cpd. with 2,2′,2″-nitrilotris(ethanol), and less than 20% (w/w)poly(oxy-1,2-ethanediyl),alpha-(undecyl)-omega-hydroxy (InternationalProducts Corporation, Burlington, N.J.), and polyoxyethylene detergents(e.g., Triton® X-100). Most preferred are SDS and LLS at a concentrationrange preferably of from about 0.005% to about 1% (w/v), about 0.005% toabout 0.1% (w/v), or about 0.005% to about 0.02% (w/v) in the finalworking solution.

The formulation further includes the solubilizing agent for helping tomaintain the components of the formulation in solution. The solubilizingagent may contain, for example, an organic solvent or an emulsifyingagent, such as that found in Fragrance No. 2141-BG, a citrus fragranceavailable from International Flavors and Fragrances (IFF) of Hazlet,N.J. Fragrances may have the additional advantage of masking the odor ofthe deactivating agent (e.g., sodium hypochlorite). Organic solventsthat may be included in the formulation include benzyl acetate, PS20 andisopropanol. Emulsifying agents that may be included in the formulationinclude polyoxyethylene sorbitan mono-palmitate (Tween® 40), lecithinand ethylene glycol distearate. In some cases, the inventors discoveredthat the wetting agent was necessary to maintain the solubilizing agentin solution when combined with the corrosion-inhibiting agent and thatthe solubilizing agent was necessary to maintain the detergent insolution when combined with the corrosion-inhibiting agent. And, whenthe formulation also include the deactivating agent, all four componentsremained in solution. When the solubilizing agent is a fragrance, suchas IFF Fragrance No. 2415-BG or 2141-BG, the preferred concentration ofthe solubilizing agent in a final working solution which contains thedeactivating agent is in a range from about 0.001% to about 20% (v/v),about 0.001% to about 2% (v/v), or about 0.002% to about 0.2% (v/v). Theconcentration of the solubilizing agent selected should be such that ithas no substantial impact on the activity and stability of thedeactivating agent and the corrosion-inhibiting agent.

In a particularly preferred formulation of the present disclosure, a6.7× concentrate is prepared having the following formulation: 600 mMsodium bicarbonate, pH 9.3+0.1% SDS (w/v)+0.05% (v/v) IFF Fragrance No.2145-BG. When the formulation further includes a deactivating agent, aparticularly preferred formulation is as follows: 0.6% (w/v) sodiumhypochlorite+90 mM sodium bicarbonate, pH 9.3+0.015% (w/v) SDS+0.0075%(v/v) IFF Fragrance No. 2145-BG. Of course, the components andconcentrations of these preferred formulations can be modified in themanner described herein, without the exercise of undue experimentation,to arrive at alternative formulations that are stable and capable ofdeactivating nucleic acids on a surface or in a solution whileminimizing the potential corrosive effect of the deactivating agentselected.

Based on our discovery that the order in which the agents are combinedcan be important to preventing the formation of precipitates or anotherwise non-homogenous formulation, a further embodiment of thepresent disclosure is directed to a method of making the above-describedformulations. This method includes the following ordered steps: (i)separately dissolving solid forms of a corrosion-inhibiting agent and awetting agent; (ii) combining together the dissolved forms of thecorrosion-inhibiting agent and the wetting agent to form a mixture; and(iii) combining together a solubilizing agent and the mixture to form aformulation comprising the corrosion-inhibiting agent, the wetting agentand the solubilizing agent, where the agents of this formulation remainsubstantially in solution at 22° C. (approximately room temperature). Ifthe solubilizing agent is provided in a solid form, it too may bedissolved prior to combining the solubilizing agent with the mixture.The deactivating agent can then be added to the formulation, where thedeactivating agent may be added directly to the formulation or it may bedissolved prior to combining it with the formulation. If water is usedto dissolve any of the solid forms of the agents, it is preferablydistilled or deionized water. For many of the formulations tested, itwas discovered that deviating from the above-ordered steps for combiningthe agents resulted in the formation of non-homogenous solutions (e.g.,the solubilizing agent was first combined with either thecorrosion-inhibiting agent or the wetting agent).

For those applications that do not require a wetting agent, wediscovered that the deactivating agent and the corrosion-inhibitingagent may be combined without substantially affecting the ability of thedeactivating agent to deactivate nucleic acids. Therefore, formulationsof the present disclosure containing corrosive deactivating agents arenot required to include a wetting agent and a solubilizing agent.

Another preferred deactivating agent of the present disclosure compriseshydrogen peroxide and metal ions, such as, for example, copper, cobalt,iron or manganese ions (e.g., cupric sulfate or cupric acetate). Forsolution-based applications in particular, we found that the metal ions(e.g., copper ions) can be stabilized in a chemical configuration thatis active with hydrogen peroxide at deactivating nucleic acids when thedeactivating agent further includes piperazine or reagents that containthe piperazine group, such as the buffer HEPES(N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid)), acetate, andlike compounds and reagents. Surprisingly, we further discovered thatpiperazine can stimulate the deactivation of nucleic acids in thepresence of hydrogen peroxide and copper ions. The hydrogen peroxide ofthis deactivating agent is preferably present at a concentration rangeof from about 0.5% to about 30% (w/v), about 1% to about 15% (w/v), orabout 1% to about 6% (w/v). Where, for example, copper sulfate is thesource of the metal ions, the preferred concentration range of coppersulfate is from about 0.1 mM to about 5 mM, about 0.5 mM to about 2.5mM, or about 1 mM to about 2.5 mM. And if piperazine is used tostimulate the deactivation of nucleic acids, the preferred concentrationrange of piperazine is from about 0.5 mM to about 250 mM, about 1 mM toabout 200 mM, or about 10 mM to about 100 mM. A preferred formulation ofthis embodiment comprises 3% (w/v) hydrogen peroxide+2 mM CuSO₄+50 mMpiperazine, pH 5.5. This deactivating agent has the advantage of beingnon-corrosive and odorless.

In a further embodiment, the present disclosure relates to a method fordeactivating nucleic acids suspected of being present on a surface. Inthis method, a first amount of a first reagent comprising a deactivatingagent is applied to the surface. Where warranted by the expectedpresence of interfering substances (e.g., organic load and/or oily filmsor residue on the surface), and to ensure adequate deactivation ofnucleic acids present on the surface, a second amount of a secondreagent comprising a deactivating agent can be applied to the surface.The first and second reagents of this method may be the same ordifferent and one or both of the reagents may comprise one of theformulations described above. In a preferred embodiment, the reagentsare removed from the surface, such as by wiping with an absorbentmaterial (e.g., a paper towel or cotton gauze), before the reagents havehad an opportunity to completely evaporate. By wiping before thereagents have completely evaporated, nucleic acids that may not havebeen chemically deactivated by the reagents can be mechanically removedby the absorbent material. Additionally, by wiping with an absorbentmaterial after the first application, other materials solubilized by thefirst reagent that might consume all or part of the deactivating agentin the second application can be removed. Therefore, in a particularlypreferred mode, there is no substantial “soak time” between the applyingand removing steps of the preferred embodiment. This means that thedelay between application of a reagent to the surface and its removaltherefrom is no more than a few minutes, preferably no more than oneminute, and, more preferably, the removal of the reagent from thesurface immediately follows its application thereto. Also, to avoid allpossible sources of contamination, it is recommended that the reagentsfor deactivating nucleic acids be applied with one gloved hand and thatremoval of the reagents be performed with another gloved hand.

To reduce the organic load on a surface prior to application of thefirst reagent, the surface may be pre-treated with an application of adetergent. Additionally, for surface applications, it is recommendedthat the surface not be cleaned with water following removal of thefirst or second reagents from the surface, as the water may containamplifiable nucleic acids or nucleic acids or other chemicals that couldinterfere with an amplification reaction.

In still another embodiment, the present disclosure relates to a methodfor deactivating nucleic acids suspected of being present in one or moreconduits using a formulation described above. The conduits may bepresent, for example, in one or more pipettes or an aspirator manifold.In this method, the formulation containing the deactivating agent isdrawn into the one or more conduits, such as by suctioning. Theformulation is then dispensed from the one or more conduits. Afterdispensing the formulation, the one or more conduits may be exposed to awash solution by drawing the wash solution into the one or more conduitsand then dispensing the wash solution from the conduits. The washsolution may be, for example, purified water or a reagent solution andis used to rinse residual amounts of the formulation from the conduits.

In yet another embodiment, the present disclosure relates to a kitcomprising, in one or more receptacles, a formulation as described abovefor use in deactivating nucleic acids. In one embodiment, if the kitincludes a deactivating agent, the deactivating agent is preferablycontained in a receptacle separate from one or more receptaclescontaining the corrosion-inhibiting agent, the wetting agent and/or thesolubilizing agent. One or more of the components of the formulation maybe provided in a pre-measured amount suitable for making a specificvolume of final solution or as a bulk powder. If pre-measured, powderforms of the component or components may be provided in packets orcapsules or as tablets to be dissolved in water before being combinedwith the other components of the formulation. The kit may furtherinclude instructions recorded in tangible form (e.g., paper, diskette,CD-ROM, DVD or video cassette) for combining the deactivating agent andthe other components of the formulation. The kit may also include one ormore reagents for performing a nucleic acid amplification reaction. Suchreagents may include one or more enzyme reagents (e.g., an RNA or a DNApolymerase) for use in amplifying a nucleic acid sequence of interest.Enzyme reagents for use in performing a transcription-basedamplification, for example, include a reverse transcriptase and an RNApolymerase (e.g., T7 RNA polymerase). Other amplification reagents mayalso be included, such as, for example, amplification oligonucleotides(e.g., primers, promoter-primers and/or splice templates), nucleotidetriphosphates, metal ions and co-factors necessary for enzymaticactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an electrophoretogram showing the results of a fixed amount ofa 71-mer, single-stranded DNA oligonucleotide reacted with varyingconcentrations of bleach following polyacrylamide gel electrophoresis(PAGE).

FIG. 2 is an electrophoretogram showing the results of a fixed amount ofa 60-mer, single-stranded DNA/RNA chimera oligonucleotide reacted withvarying concentrations of bleach following PAGE. The RNA consisted of2′-O-methyl ribonucleotides.

FIG. 3 is comprised of two electrophoretograms showing the results of afixed amount of a 71-mer, single-stranded DNA oligonucleotide reactedwith varying concentrations of dichloroisocyanurate or bleach,respectively, following PAGE.

FIG. 4 is an electrophoretogram showing the results of a fixed amount ofa 71-mer, DNA oligonucleotide reacted with varying concentrations ofhydrogen peroxide alone or in combination with a fixed concentrationcupric sulfate following PAGE.

FIG. 5 is an electrophoretogram showing the results of a fixed amount ofa 71-mer, DNA oligonucleotide reacted with varying concentrations ofhydrogen peroxide and a fixed amount of cupric sulfate following PAGE.

FIG. 6 is comprised of two electrophoretograms showing the results of afixed amount of a 71-mer, single-stranded DNA oligonucleotide reactedwith varying concentrations of bleach in the presence or absence of NALCfollowing PAGE.

FIG. 7 is an electrophoretogram showing the results of a fixed amount ofa 71-mer, single-stranded DNA oligonucleotide reacted with varyingconcentrations of hydrogen peroxide and a fixed amount of cupric sulfatein the presence or absence of a fixed amount of NALC or human serumfollowing PAGE.

FIG. 8 is a graph showing the results of a real-time amplification afterreacting a target nucleic acid with varying concentrations of bleach ina pure system.

FIGS. 9A-9H show assay results for nucleic acid deactivation by bleach.

FIGS. 10A-10F show real-time transcription-mediated amplification (TMA)results for nucleic acid deactivation by bleach in the presence oforganic load.

FIGS. 11A-11D show PAGE results for nucleic acid deactivation by bleachin the presence of organic load.

FIG. 12 shows PAGE results illustrating scavenging effects of EnzymeDilution Buffer on DCC and bleach.

DETAILED DESCRIPTION

The present disclosure is directed in part to formulations, methods andkits which are useful for deactivating nucleic acids. Theseformulations, methods and kits are described above and in the examplesand claims which follow. In addition, the examples describe screeningmethods for selecting formulations of the present disclosure which areuseful for deactivating nucleic acids on work surfaces, laboratoryequipment and/or in solution, or which could be used as, for example,disinfectants. Such formulations may also be useful for deactivatingbiological molecules, like proteins and lipids. The examples furtherconsider the effect of a number of exemplary formulations in both pre-and post-amplification applications.

EXAMPLES

The examples set forth below illustrate but do not limit the disclosure.

Example 1 Effect of Various Concentrations of Bleach on Visualization ofDNA by Gel Electrophoresis

An experiment was conducted in which a 71-mer DNA oligonucleotide wasreacted with various concentrations of Ultra Clorox® Bleach (The CloroxCompany, Oakland, Calif.) at a concentration of 6.15% (w/v) sodiumhypochlorite, and the reaction products were analyzed usingpolyacrylamide gel electrophoresis (PAGE). Ten samples were prepared bymixing 2 μL of the DNA oligonucleotide, at a concentration of 173 μg/mL,with distilled water in sample vials before adding varyingconcentrations of bleach to bring the total volume of each sample to 20μL. The samples were mixed by vortexing for about 10 seconds and thenprovided with 20 μL of a 2×TBE-Urea sample buffer containing 180 mM Trisbase, 180 mM boric acid, 4 mM ethylenediaminetetraacetic acid (EDTA), pH8.0 (Invitrogen Corporation, Carlsbad, Calif.; Cat. No. LC 6876),bringing the total volume of each sample to 40 μL. The samples wereagain mixed by vortexing for about 10 seconds. Final bleachconcentrations in the samples ranged from 0 to 50% bleach, as set forthin Table 1 below. A 10 μL aliquot of each sample was loaded into one ofthe 10 lanes of a 10% polyacrylamide TBE-Urea gel, and the gel was runfor 40 minutes at 180 V. When the run was completed, the gel was removedfrom its cast, contacted with 100 mL of a SYBR® Green I nucleic acid gelstain (Molecular Probes, Eugene Oreg.; Cat. No. S7563) diluted 1/10,000with distilled water, and mixed at 10 rpm for 30 minutes. Afterstaining, the gel was photographed using a Chemilmager™ System 4400(Alpha Innotech Corporation, San Leandro, Calif.). The separatedproducts stained on a gel are commonly referred to as bands. A copy ofthe resulting electrophoretogram is presented in FIG. 1.

TABLE 1 Bleach Concentrations Lane % Bleach 1 0 2 0.01 3 0.03 4 0.1 50.3 6 1 7 3 8 10 9 20 10 50

From the results illustrated in the electrophoretogram of FIG. 1, it canbe seen that the last visible band appears in lane 3. These resultssuggest that between 0.03% (0.25 mM) and 0.1% (0.82 mM) sodiumhypochlorite was needed to substantially alter the DNA present in thesamples. From the DNA oligonucleotide concentration indicated above, itwas determined that the nucleotide concentration in the samples was 0.52mM. Thus, the sodium hypochlorite to nucleotide molar ratio wasapproximately 1:1, suggesting that about one mole sodium hypochloritereacted with about one mole nucleotide. Another similar experimentcomparing incubation times of 0 to 20 minutes showed no changes in theappearance of oligonucleotide bands over the time course of hypochloriteincubation, suggesting the reaction rate is rapid.

Example 2 Effect of Various Concentrations of Bleach on Visualization ofa DNA/RNA Chimera by Gel Electrophoresis

The experiment of Example 1 was repeated, substituting a 60-mer DNA/RNAchimeric oligonucleotide at a concentration of 200 μg/mL for the DNAoligonucleotide of that experiment. The RNA of the chimera consisted of2′-O-methyl ribonucleotides. A copy of the resulting electrophoretogramappears in FIG. 2 and shows that most of the oligonucleotide banddisappeared at 0.1% bleach, again about a 1:1 molar ratio of sodiumhypochlorite to nucleoside. The concentrations of bleach used in thevarious lanes of the gel of this experiment are the same as thosedescribed in the experiment of Example 1.

Example 3 Effect of Various Concentrations of Bleach on Visualization ofDNA by Gel Electrophoresis

Dichloroisocyanuric acid, sodium salt (DCC) (Sigma-Aldrich, Milwaukee,Wis.; Prod. No. 21, 892-8) and Ultra Clorox® Bleach (6.15% (w/v) sodiumhypochlorite) were examined at varying available chlorine concentrationsin this experiment for their comparative abilities to react with nucleicacid. The chlorine concentrations tested are set forth in Table 2 below.In all other aspects, including the use of the 71-mer DNAoligonucleotide, this experiment was identical to the experimentdetailed in Example 1.

TABLE 2 Chlorine Concentrations Lane Chlorine (mM) 1 0 2 0.8 3 0.24 40.8 5 2.4 6 8 7 24 8 80 9 160 10 400

The results of this experiment are illustrated in FIG. 3 and indicatethat pure DCC causes the disappearance of the DNA band at lowerconcentrations than the bleach solution at the same chlorineconcentration.

Example 4 Effect of Various Concentrations of Hydrogen Peroxide andHydrogen Peroxide Plus Cupric Sulfate on Visualization of DNA by GelElectrophoresis

In this experiment, a 71-mer DNA oligonucleotide present at aconcentration of 53 μg/mL was reacted with various concentrations of 30%(w/v) hydrogen peroxide (Fisher Scientific, Tustin, Calif.; Cat. No.BP2633-500) and 30% (w/v) hydrogen peroxide plus cupric sulfate(Sigma-Aldrich, Milwaukee, Wis.; Prod. No. 45, 165-7), and the reactionproducts were analyzed using polyacrylamide gel electrophoresis (PAGE).Ten samples were prepared in the manner indicated in Table 3 below, withthe DNA and water being combined prior to adding 30% (w/v) hydrogenperoxide (8.8 M) and/or 1 mM cupric sulfate. The remaining proceduraldetails of this experiment are the same as those set forth in Example 1.The concentration of peroxide in each lane is set forth in Table 4below.

TABLE 3 Sample Mixtures Components (μL) DNA CuSO₄ H₂O₂ H₂O Sample 1 2 00 18 Number 2 2 2 0 16 3 2 0 16 2 4 2 2 0.33 15.7 5 2 2 1 15 6 2 2 2 147 2 2 4 12 8 2 2 8 8 9 2 2 12 4 10 2 2 16 0

TABLE 4 Hydrogen Peroxide Concentrations Lane % H₂O₂ (w/v) 1 0 2 0 3 244 0.5 5 1.5 6 3 7 6 8 12 9 18 10 24

The resulting electrophoretogram appears in FIG. 4 and indicates thatthe peroxide and the cupric sulfate do not independently cause thedisappearance of the DNA oligonucleotide bands at the indicatedconcentrations. However, the electrophoretogram does appear todemonstrate that mixtures of peroxide and cupric sulfate are effectiveat causing the disappearance of the DNA oligonucleotide bands at allconcentrations tested. This suggests that the cupric sulfate mayfunction as a catalyst for the peroxide in the degradation of nucleicacids.

Example 5 Effect of Various Concentrations of Hydrogen Peroxide in thePresence of Cupric Sulfate on Visualization of DNA by GelElectrophoresis

The experiment of Example 4 was repeated using lower concentrations ofthe hydrogen peroxide component and 100 μM cupric sulfate in all lanesof the gel. The final concentration of peroxide in each lane of the gelis set forth in Table 5 below.

TABLE 5 Peroxide Concentrations Lane % H₂O₂ (w/v) 1 0 2 0.0005 3 0.005 40.01 5 0.02 6 0.04 7 0.1 8 0.2 9 0.4 10 1

A copy of the resulting electrophoretogram appears in FIG. 5 and showsno band at 0.2% (w/v) hydrogen peroxide and only a faint band at 0.1%(w/v) hydrogen peroxide.

Example 6 Effect of NALC Upon Reaction of Bleach with DNA

Bleach is known to react with a variety of organic materials. Thesematerials may thus interfere with the deactivation of nucleic acids byreacting with and consuming the bleach. The presence of these organicmaterials thus constitutes an “organic load” that must be compensatedfor by the presence of sufficient bleach to react with both the DNA andthe organic materials. In this experiment, the scavenging effect ofN-acetyl-L-cysteine (NALC), an organic load compound (i.e., a compoundthat may be expected to consume bleach), was examined in the presence ofvarying concentrations of Ultra Clorox® Bleach. NALC is a reducing agentfound in some enzyme reagents intended for use in amplificationreactions. Two sets of 10 samples were prepared in this experiment, eachsample containing 2 μL of a 71-mer DNA oligonucleotide at aconcentration of 173 μg/mL. The first set of samples contained no NALC,while each sample of the second set of samples contained 16 μL NALC at aconcentration of 11.4 mg/mL. The samples were prepared by firstproviding the DNA and NALC (if any) to sample vials and mixing thesamples containing NALC by vortexing for about 10 seconds. The bleachwas then added to both sets of samples at varying concentrations, alongwith distilled water, to bring the total volume of each sample to 20 μL.The samples were mixed by vortexing for about 10 seconds before adding20 μL of a 2×TBE-Urea sample buffer (Invitrogen Corporation; Cat. No. LC6876), bringing the total volume of each sample to 40 μL. The sampleswere again mixed by vortexing for about 10 seconds. Final bleachconcentrations in the samples ranged from 0% to 50% bleach, as set forthin Table 6 below. A 10 μL aliquot of each sample was loaded into one of10 lanes of a 10% polyacrylamide TBE-Urea gel, a separate gel beingprovided for each of the two sets of samples, and the gels were run for40 minutes at 180V. When the runs were completed, the gels were removedfrom their casts, contacted with 100 mL of a SYBR® Green I nucleic acidgel stain (Molecular Probes; Cat. No. S7563) diluted 1/10,000 withdistilled water, and mixed at 10 rpm for 30 minutes. After staining, thegels were photographed using a Chemilmager™ System 4400, and a copy ofthe resulting electrophoretogram is presented in FIG. 6.

TABLE 6 Bleach Concentrations Lane % Bleach 1 0 2 0.01 3 0.03 4 0.1 50.3 6 1 7 3 8 10 9 20 10 50

From the results illustrated in the electrophoretograms of FIG. 6, itcan be seen that the last clearly visible band appears in lane 3 (0.03%(v/v) bleach) of the gel having samples containing no NALC and in lane 8(10% (v/v) bleach) of the gel having samples containing NALC. Theseresults indicate that the concentration of bleach needed to cause thedisappearance of the DNA bands is affected by the presence of NALC,which likely competes with the DNA for reaction with bleach.

Example 7 Effect of NALC and Human Serum Upon Reaction of HydrogenPeroxide and Cupric Sulfate Mixture with DNA

In this experiment, the effect of NALC and human serum upon the reactionof various concentrations of hydrogen peroxide and cupric sulfate withDNA was examined. A set of 11 samples was prepared, each samplecontaining 2 μL of a 71-mer DNA oligonucleotide at a concentration of 53μg/mL. Other components of the samples included 100 μM cupric sulfate,30% (w/v) hydrogen peroxide, and NALC at a concentration of 11.4 mg/mL.The amount of each component in the sample vials is set forth in Table 7below. The samples were prepared by combining all sample components,except the hydrogen peroxide, in sample vials and mixing by vortexingfor about 10 seconds. After mixing, the hydrogen peroxide was added tothe samples at varying concentrations, bringing the total volume ofsample 1 to 20 μL and samples 2-11 to 22 μL and giving the finalconcentrations indicated in Table 8 below. The samples were again mixedby vortexing for about 10 seconds before adding 20 μL of a 2×TBE-Ureasample buffer (Invitrogen Corporation; Cat. No. LC 6876), bringing thetotal volume of sample 1 to 40 μL and samples 2-11 to 42 μL. Theremainder of the procedure and sources of the reagents were identical tothat set forth in Example 6 above. A copy of the resultingelectrophoretogram is presented in FIG. 7.

TABLE 7 Sample Mixtures Components (μL) Human DNA CuSO₄ H₂O₂ NALC SerumH₂O Sample 1 2 0 0 0 0 18 Number 2 2 2 1 0 0 17 3 2 2 4 0 0 14 4 2 2 120 0 6 5 2 2 1 4 0 13 6 2 2 4 4 0 10 7 2 2 12 4 0 2 8 2 2 0 0 4 14 9 2 21 0 4 13 10 2 2 4 0 4 10 11 2 2 12 0 4 2

TABLE 8 Peroxide Concentrations Lane % H₂O₂ (w/v) 1 0 2 1.36 3 5.45 416.36 5 1.36 6 5.45 7 16.36 8 0 9 1.36 10 5.45 11 16.36

The results illustrated in the electrophoretograms of FIG. 7 show thatNALC and serum interfere with the reaction of the hydrogen peroxide andcupric sulfate mixture with DNA. Thus, these results demonstrate thatthe amount of hydrogen peroxide needed to cause the disappearance of theDNA bands is affected by the presence of NALC and human serum, whichlikely compete with the DNA for reaction with bleach.

Example 8 Reaction of Nucleic Acids with Bleach in a Pure System

This experiment was conducted to evaluate the ability of various bleachconcentrations to deactivate purified ribosomal RNA derived fromNeisseria gonorrhoeae (“target”) in a pure system. Eight sample tubeswere initially set up to contain 4 μL of target-containing water and 4μL of bleach in the concentrations indicated in Table 9. For sampletubes 6 and 8, 4 μL of water was used in place of a bleach solution).The bleach used in this experiment was Ultra Chlorox® Bleach (6.15%(w/v) sodium hypochlorite). After set up, the contents of the sampletubes were incubated for 5 minutes at room temperature.

TABLE 9 Bleach Concentrations Initial Target Initial Bleach Final BleachSample Concentration Concentration Concentrations Tube (copies/μL) %(v/v) % (v/v) 1 10⁸ 40 20 2 10 5 3 4 2 4 1 0.5 5 0.4 0.2 6 0 0 7 0 40 208 0 0

Following the room temperature incubation, 392 μL of water (chilled onice) was added to each sample tube. The samples then were analyzed by areal-time Transcription-Mediated Amplification (TMA) assay. In theassay, amplification reaction mixtures were prepared by combining a 4 μLaliquot from each sample tube with 300 μL of an Amplification Reagent(44.1 mM HEPES, 2.82% (w/v) trehalose, 33.0 mM KCl, 9.41 mM rATP, 1.76rCTP, 11.76 rGTP, 1.76 mM UTP, 0.47 mM dATP, 0.47 mM dCTP, 0.47 mM dGTP,0.47 mM dTTP, 30.6 mM MgCl₂, 0.30% (v/v) ethanol, 0.1% (w/v) methylparaben, 0.02% (w/v) propyl paraben, and 0.003% (w/v) phenol red) at pH7.7 and spiked with 25.6 pmol of a T7 promoter-primer and 20.0 pmol of anon-T7 primer for amplifying a region of the target following aTranscription-Mediated Amplification (TMA) procedure (see Kacian et al.,U.S. Pat. No. 5,399,491) and 80 pmol of a molecular beacon probe fordetecting the resulting amplicon in real-time (see Tyagi et al.,“Detectably Labeled Dual Conformation Oligonucleotide Probes, Assays andKits,” U.S. Pat. No. 5,925,517). The probes and primers of thisexperiment were synthesized on an Expedite™ 8909 Nucleic AcidSynthesizer (Applied Biosystems, Foster City, Calif.) using standardphosphoramidite chemistry. See, e.g., Caruthers et al., Methods inEnzymology, 154:287 (1987). The molecular beacon probes were synthesizedto include interacting CyTM5 and BHQTM dyes using Cy5-CE phosphoramidite(Glen Research Corporation, Sterling, Va.; Cat. No. 10-5915-90) and3′-BHQ-2 Glycolate CPG (BioSearch Technologies, Inc., Novato, Calif.;Cat. No. CG5-5042G-1).

Amplification reaction mixtures were then set up in a 96-wellMicrotiter® plate (Thermo Labsystems, Helsinki, Finland; Cat. No.9502887) in replicates of three, each well containing 75 μL of a lightmineral oil and 75 μL of the amplification reaction mixture. The plateswere covered with ThermalSeal sealing film (Sigma-Aldrich Co., St.Louis, Mo.; Product No. Z36, 967-5) and incubated in a Solo HTMicroplate Incubator (Thermo Electron Corporation; Milford, Mass.) for15 minutes at 62° C. to permit hybridization of the promoter-primer tothe target, followed by a second 15 minute incubation in the Solo HTMicroplate Incubator at 42° C. After incubating the contents of theplate, a multi-channel pipettor was used to add 25 μL of an EnzymeReagent (50 mM N-acetyl-L-cysteine (NALC), 58 mM HEPES, 3.03% (w/v)trehalose, 10% Triton® X-100 detergent, 1.04 mM EDTA, 20% (v/v)glycerol, 120 mM KCl, 120 RTU/μL Moloney murine leukemia virus (“MMLV”)reverse transcriptase, and 80 U/μL T7 RNA polymerase, where one “unit”of activity is defined as the synthesis and release of 5.75 fmol cDNA in15 minutes at 37° C. for MMLV reverse transcriptase, and the productionof 5.0 fmol RNA transcript in 20 minutes at 37° C. for T7 RNApolymerase) at pH 7.0 to each sample Immediately after each set ofEnzyme Reagent additions, the contents of the reaction wells were mixedby stirring with the corresponding pipette tips held by the pipettor. Tomeasure the formation of amplicon in real-time, the plate wastransferred to a Fluoroskan Ascent microplate fluorometer (ThermoElectron Corporation; Product No. 5210470) and incubated for 60 minutesat 42° C. Fluorescence from the reaction wells was measured in 30 secondincrements using a 639 nm excitation filter and 671 nm emission filter.

The results of this experiment are reported in the graph of FIG. 8,which plots relative fluorescent units (RFU) on the y-axis and time inminutes on the x-axis. The results show that even at 0.2% bleach, thelowest bleach concentration tested, the target nucleic acid in this puresystem was deactivated, such that it could not be detectably amplified.Detectable amplification in this experiment would have been RFU valuemore than two-fold the background RFU value (sample tube 8) in a 60minute amplification period.

Example 9 Further Characterization of Nucleic Acid Deactivation in aPure Bleach System

Several formulations were tested for efficacy in deactivating nucleicacids using multiple assays.

A. Real-Time TMA Results

Neisseria gonorrhoaea (Ngo) ribosomal RNA (rRNA) was reacted with 0-20%commercial bleach, where the lowest bleach concentration was 0.2%, in apure system and reaction products were analyzed by real-time TMA assays(see Example 8). Even at the lowest bleach concentration the rRNA wasinactivated within the limits of sensitivity of the real-time assay(FIG. 9A).

Chlamydia trachomatis (Ctr) rRNA also was reacted with 0-20% bleach,where the lowest bleach concentration was 0.016%, in a pure system andreaction products were analyzed by real-time TMA assays. The lowestbleach concentration also inactivated the rRNA (FIG. 9B).

B. Capillary Electrophoresis Results

Ribosomal RNA was reacted with bleach in solution and products wereanalyzed by capillary electrophoresis. An Agilent 2100 Bioanalyzer wasutilized to characterize nucleic acids exposed to deactivationsolutions. In a 10 μL total reaction, the following were added (inorder): (a) Milli-Q H₂O or buffer, (b) an indicated amount of reagent(e.g., —OCl from bleach or H₂O₂), and (c) 0 nM (blank) or 150 nM (718μM=470 ng/μL nt) Mycobacterium tuberculosis (Mtb) rRNA or 15 nM (71.8μM=47.0 ng/μL nt) Mtb rRNA. The reactants were incubated for 10 min atroom temperature (ca. 23° C.), and 90 μL 1 mM sodium ascorbate (900 μMfinal) then was added. As in the LabChip® protocol (AgilentTechnologies, Inc.; Palo Alto, Calif.), the RNA ladder was denatured at70° C. for 2 min, and then 1 μL of each reaction was loaded into wellson RNA 6000 Nano LabChip® or Pico LabChip® (Agilent Technologies, Inc.;Palo Alto, Calif.) containing 5 μL sample buffer. The components weremixed and the assay Prokaryote Total RNA was run in the Bio Sizingprogram (Agilent Technologies, Inc.; Palo Alto, Calif.).

Results from the capillary electrophoresis analysis showed a 1:1 ratioof hypochlorite-to-rRNA nucleotide substantially eliminated rRNA peaks(FIG. 9C to 9F). A time course of the reaction between rRNA and bleachalso was performed (FIGS. 9G and 9H). The reaction with both the 165 and23S subunits is very fast, essentially over within 1 min, withpseudo-first order rate constants for the decay of rRNA approaching atleast 0.02 s⁻¹.

C. Conclusions

Reaction of bleach (hypochlorite) with nucleic acids in a pure systemwas rapid and essentially complete at a 1:1 ratio of hypochlorite tonucleoside. These data suggested that any observed lack ofdecontamination of nucleic acids in the laboratory using bleach was notdue to an inherently slow reaction of hypochlorite with the nucleicacids or the need for a high molar excess of bleach over the nucleicacids.

Example 10 Further Characterization of Nucleic Acid Deactivation byBleach in the Presence of Organic Load

Effects of N-acetyl-L-cysteine (NALC), an organic load material, on thereaction between bleach and oligonucleotides were characterized by PAGEin Example 6. Presented hereafter is a characterization of the effectsof NALC and other organic load materials on the reaction betweenoligonucleotides and bleach using PAGE and other characterizationmethods.

A. Real-Time TMA Results

Ribosomal RNA was reacted with bleach in the presence of differentamounts of various organic load materials. The ability of this RNA to beamplified was then tested using real-time TMA. Organic load materialsincluded Amplification, Hybridization, Enzyme and Selection Reagentsfrom the Aptima Combo 2® Assay kit (Catalog No. 1032; Gen-ProbeIncorporated; San Diego, Calif.), and mixtures thereof, urine transportmedium (UTM; Catalog No. 1040 Aptima Combo 2® Assay Urine SpecimenCollection Kit for Male and Female Urine Specimens; Gen-Probe), swabtransport medium (STM), KOVA-Trol™ (Hycor Biomedical Inc.; Garden Grove,Calif.), bovine serum albumin (BSA), lithium lauryl sulfate (LLS) andhuman plasma. Of these compounds, UTM and Enzyme Reagent were mosteffective at interfering with reaction of the bleach with RNA. In oneexperiment, 20% commercial bleach was required to overcome the effectsof UTM, which is in contrast to the very rapid and complete reaction ofrRNA with 0.016% bleach in the absence of organic load materials (FIGS.10A-10F).

B. PAGE Results

PAGE was performed using a procedure similar to that disclosed inExample 1. Briefly, a known amount of a 71-mer oligonucleotide wasincubated with a formulation having a known concentration of candidatereagent. A 1× volume of 2×TBE-urea loading buffer (180 mM Tris, 180 mMboric acid, 4 mM EDTA, pH 8.0) was added to the mixture solution andvortexed for 10 seconds. Ten microliters of sample was loaded in eachlane of a 10% polyacrylamide TBE-Urea gel. The gel was run in 1×TBErunning buffer at 180 V for 35 to 40 minutes depending on the length ofoligonucleotide. The gel then was removed from the cast and stained in1/10,000 SYBr Green I dye solution for 20 minutes. The stained gel wasimaged using a Chemilmager™ 4400.

Oligonucleotides were reacted with bleach in the presence of variousconcentrations of organic load compounds, and reaction products wereanalyzed by PAGE. Serum, Amplification Reagent and the NALC in EnzymeDilution Buffer interfered with the reaction of bleach with theoligonucleotide (FIGS. 11A-11D).

C. RP-HPLC Results

Reverse phase (RP) HPLC was utilized to characterize nucleic acidsexposed to deactivation solutions using standard procedures.Specifications for the HPLC apparatus and methodology utilized were asfollows. A Zorbax® Eclipse XDB C-8 Reverse Phase Column (AgilentTechnologies, Inc.; Palo Alto, Calif.) having a 4.6 mm internal diameterand a 15 cm length was utilized. Triethyl ammonium acetate(TEAA)/acetonitrile was utilized as the mobile phase, where Buffer Acontained 0.1M TEAA and Buffer B contained 100% acetonitrile. A gradientof 5%-100% Buffer B was utilized in a time interval of 15 minutes at aflow rate of 0.5 mL/min. 50 μL oligonucleotide samples having an opticaldensity of 2.0 OD (oligonucleotide 26mer=10 nM) were injected on thecolumn and column output was detected at a wavelength of 254 nm.

Reaction of bleach with a 26mer DNA oligomer in the presence of NALC andsubsequent chromatography using RP-HPLC revealed that NALC interferedwith the reaction of bleach with DNA. These results confirmed PAGEfindings in Example 6.

D. Conclusions

Materials that effectively interfered with the reaction of bleach withnucleic acids were Urine Transport Medium (UTM) and the NALC in EnzymeDilution Buffer (EDB). Materials that moderately interfered with thereaction of bleach with nucleic acids were Swab Transport Medium (STM),Hybridization Reagent, Amplification Reagent and human serum. Materialsthat weakly interfered with the reaction of bleach with nucleic acids(or not at all) were Selection Reagent, Aptima Combo 2® Assay TargetCapture Reagent, lithium lauryl sulfate and KOVA-Trol™. From thisanalysis, it was determined that organic load material, especiallymaterials containing primary amine and sulfhydryl groups, reacted withbleach and consumed it so that it was not all available to deactivatethe nucleic acids. Loss of decontamination power of bleach at lowerconcentrations was not due to slow reaction rates or the need for excesshypochlorite over nucleotides, but rather consumption of bleach by othercompounds.

Example 11 Further Screens of Alternative Formulations and Conditions

Alternative formulations to bleach, such as solutions containingdichloroisocyanuric acid (DCC) or hydrogen peroxide and copper ions,were characterized in Examples 3 and 4 by PAGE. These and additionalalternative formulations were characterized by PAGE and other assays asdescribed hereafter.

A. PAGE Results

A 71-mer oligonucleotide was reacted with various candidate compoundsand the products were analyzed using PAGE. Solutions containing DCC orhydrogen peroxide with copper sulfate were tested, among otherformulations. As shown in Example 3, DCC, which is less corrosive thanbleach, was as effective as bleach for deactivating the oligonucleotide,if not more so. The effects of scavengers including Enzyme DilutionBuffer (EDB) and serum on DCC were also tested and compared with theireffects on bleach. Similar effects were observed as shown in FIG. 12(results are for EDB; serum results not shown). As shown in Examples 4,and 7, a solution containing hydrogen peroxide and copper sulfate, whichwas odorless and non-corrosive, was reasonably effective at (1) changingoligonucleotide migration or oligonucleotide band retention, and (2)overcoming the effects of organic load.

Other candidate solutions were characterized by incubating them witholigonucleotide and analyzing the resulting reaction products by PAGE.The following reagents exhibited little or no changes to nucleic acidmigration or band intensity in this assay: (1) peroxymonosulfate (KHSO₅)with or without copper sulfate; (2) perborate; (3) percarbonate; (4)hydrogen peroxide with KBr; and (5) NucleoClean™ (ChemiconInternational, Inc.; Temecula, Calif.).

B. RP-HPLC Results

The RP-HPLC retention shift assay (described previously) was used toscreen several bleach alternative candidates in the presence or absenceof organic load material (NALC). A summary is provided in Table 10 belowof the efficacy of the alternative formulations tested as compared to10% bleach, where “=” is roughly equivalent, “<” is less effective and“>” is more effective.

TABLE 10 Effectiveness of Bleach Alternative Formulations BleachAlternative Reagent Organic Load Effectiveness NaBr/NaOCl 70 mM NALC >KBr/peroxomonosulfate 70 mM NALC < ClO2 70 mM NALC < 10% bleach/peroxide70 mM NALC = Citric Acid None < citric acid/peroxide None < 10%bleach/citric acid/peroxide 70 mM NALC = 10% bleach/peroxide/sodium 70mM NALC = hydroxide phosphoric acid/peroxide None < peroxide/CuSO₄ None=, > peroxide/CuSO₄ 70 mM NALC =, > peroxide/CuSO4/phosphoric 70 mM NALC< acid 10% bleach/peroxide 70 mM NALC = Citric Acid None <Formulations comprising (a) NaBr/NaOCl or (b) peroxide/CuSO₄ were aseffective or more effective for deactivating nucleic acids as comparedto bleach alone under the conditions of this experiment.

C. Capillary Electrophoresis Results

Ribosomal RNA was reacted with various candidate formulations insolution and the products were analyzed using a capillaryelectrophoresis assay. In the assay, 1 mM dichloroisocyanurate (DCC) and17.5 mM peroxymonosulfate (Virkon® S; DuPont Animal Health Solutions,United Kingdom), tested separately, substantially eliminated peakscorresponding to 0.72 mM rRNA oligonucleotide. In situ-generated Cl₂ (10mM peroxymonosulfate+20 mM KCl) partially eliminated 72 μM rRNAoligonucleotide. Tested separately, (a) in situ-generated Br₂ (10 mMperoxymonosulfate+20 mM KBr), (b) between 10 and 100 μMdichloro-hydantoin or dibromo-hydantoin, (c) between 10 and 100 μMhypobromite, and (d) 10 mM peroxymonosulfate+metal ions (1 mM Cu²⁺, 1 or10 mM Fe²⁺) substantially eliminated 72 μM rRNA oligonucleotide.

D. Real-time TMA Results

Ribosomal RNA was reacted with various compounds in solution, and theability of the RNA to be amplified was then tested using the real-timeTMA assay described in Example 8. The efficacies of certain formulationsare described hereafter.

Virkon® S (Peroxymonosulfate).

The nucleic acid was reacted with a 2.5% Virkon® S solution (about 8.7mM peroxymonosulfate), which was a substantially lower concentrationthan the organic load included in the reaction (Enzyme Dilution Buffer(EDB) or Urine Transport Medium (UTM) here). Thus, 2.5% Virkon® Ssolution did not substantially inactivate the nucleic acid target in thepresence of 5 μL EDB or UTM.

DCC.

An 83 mM DCC solution, which was determined as approximately equivalentto 10% bleach, inactivated target in the presence of EDB.

Peroxymonosulfate/KBr.

Target rRNA in the presence of UTM was inactivated with 0.25 Mperoxymonosulfate/0.25 M KBr. Other ratios tested were not as effective,and an optimum ratio is determined by varying the ratio in additionalruns of the assay. At 0.25 M of each component, intensive coloration andodor were observed (due to the Br₂), and after addition to UTM/Targetmix, a residue formed. The residue dissolved upon a 50× dilution inwater. The stability of this formulation may be characterized further byvarying reaction conditions in additional runs of the assay. Ifformulations including these components are found to have limitedstability, they can be provided in dry powder formulations and thesolutions can be prepared shortly before use.

Perborate and Percarbonate.

Perborate was not sufficiently soluble at concentrations useful insolution. Percarbonate was soluble to 880 mM (roughly the equivalent of3% peroxide). When combined with copper(II), percarbonate at thisconcentration reacted with nucleic acid essentially with the efficacy of3% hydrogen peroxide. Percarbonate evolved oxygen quite readily whenmixed with copper(II), however, indicating the stability of the activereagents would require additional testing by the assay. Also, whenpercarbonate was combined with copper(II)/piperazine, a yellow residueformed. Enhanced activity was observed in solution (as with hydrogenperoxide/copper(II)/piperazine), but the solution characteristics werenot ideal (lower solubility, foamy). Accordingly, while the percarbonatesolutions were effective nucleic acid deactivators, the solutionproperties were less favorable than hydrogen peroxide formulations.Provision of the components in dry form to prepare solutions just priorto use would overcome some of these disadvantages.

From these results, the compounds that were especially effective (atappropriate concentrations) included bleach+peroxide, KHSO₅+KBr, DCC andperoxide+UTM. Compounds that were not as effective under the particularconditions of the experiments include 15% peroxide alone;peroxide+potassium, sodium or iron ions; 5 mM bromo- or chloro-hydantoinand KMnO₄. The effectiveness of peroxide+copper was not determined atthe time of these studies since the corresponding control failed (i.e.,the reaction mix itself inhibited TMA). It also was determined 1 mMCuSO₄/3% H₂O₂ inactivated rRNA oligonucleotide to a greater degree than1 mM CuBr₂/3% H₂O₂, CuCl₂/3% H₂O₂, or Cu(NO₃)₂/3% H₂O₂. Additionally, 1mM Cu(OAc)₂/3% H₂O₂ inactivated rRNA to a greater degree than 1 mMCuSO₄/3% H₂O₂.

Results from the analytical methods described herein are summarized inthe following Table 11 below. In the Table, “+” indicates the compoundwas deactivating; “−” indicates the compound was not substantiallydeactivating under the conditions and by the methods used; “*” indicatesequivocal results were obtained and further results can be obtained byrepeating the assay at the conditions shown; no notation indicates theconditions were not examined by the indicated assay.

TABLE 11 Effectiveness of Deactivating Reagents Compound Bioanalyzer TMAHPLC, MS PAGE HOCl + + + + HOBr + Cl₂ (from − peroxymonosulfate + KCl)Br₂ (from + + − peroxymonosulfate + KBr) I₂ (from − peroxymonosulfate +KI) DCC (ACT 340 PLUS + + + 2000 ® disinfectant) DCC + +halo-hydantoins + + HOCl + tertiary amines − NaBr + NaOCl + ClO₂ − H₂O₂− − − H₂O₂ + metal ions + + H₂O₂ + metal ions + + ascorbate H₂O₂ + HOCl− − H₂O₂, acidic − H₂O₂, acidic + metal ions + H₂O₂, acidic + HOCl (two− step addition) H₂O₂, basic + HOCl (two − step addition) H₂O₂ + KBr orNaCl − − Chloramine-T − peracetic acid (Peroxill − 2000) perborate − −percarbonate * − Virkon ® S solution +/− − (peroxymonosulfate)peroxymonosulfate + + peroxymonosulfate + − Cu(II) DNA AWAY ™ solution −DNA-OFF ™ cleansing − solution DNAZAP ™ + decontamination solutionNucleoClean ™ − − decontamination solution Citric acid −

In this Table, DNA AWAY™ is an alkali hydroxide solution (MolecularBioProducts, Inc., San Diego, Calif.; Cat. No. 7010), DNAZap™ is a pairof PCR DNA degradation solutions (Ambion, Inc., Austin, Tex.; Cat. No.9890), DNA-OFF™ is a non-alkaline cleaning solution (Q-biogene, Inc.,Irvine, Calif.; Cat. No. QD0500), and NucleoClean™ is a PCRdecontamination solution (Chemicon International, Temecula, Calif.; Cat.No. 3097S). These results showed bleach (at reduced levels),dichloroisocyanurate (DCC), H₂O₂/Cu(II), peroxymonosulfate,peroxymonosulfate/KBr (generates Br₂) and hypobromite displayedespecially potent nucleic acid deactivation activity in solution.

Example 12 Further Characterization of Nucleic Acid DeactivationFormulations and Methods in a Nucleic Acid Amplification Procedure

Multiple formulations and various methods of applying them werecharacterized for nucleic acid deactivation efficacy in an Aptima Combo2® Assay (described hereafter) and associated components. Following is alist of materials utilized for the assay and characterization process:

Amplification Reagent

Amplification Reconstitution Solution

Target Capture Reagent

Target Capture Reagent B

CT Positive Control

GC Positive Control

Oil Reagent

Wash Buffer

Urine Transport Media (UTM)

Swab Transport Media (STM)

Enzyme Reagent

Enzyme Reconstitution Solution

CT rRNA

GC rRNA

KOVA-Trol™ (Normal)

Probe Reagent

Probe Reconstitution Solution

Selection Reagent

Detection Reagent I

Detection Reagent II

Endocervical swabs

Household liquid bleach (Chlorox®)

Dichloroisocyanurate (DCC)

Household hydrogen peroxide, 3% U.S.P. (H₂O₂)

Cupric sulfate (Cu(II))

Peroxymonosulfate (KHSO₅)

Following is a description of several analytical processes employed forthe characterization procedures.

A. Preparation of Positive and Negative Amplification Reactions

Oil reagent (200 microliters) was added to 80 reaction tubes (12×75 mm)4.2×10¹⁰ copies of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae(GC) rRNA were spiked into 3.15 mL of reconstituted AmplificationReagent. Seventy-five microliters (1×10⁹ copies (˜2.5 ng)) of thisspiked Amplification Reagent was added to 40 of the reaction tubes(positive samples). Seventy-five microliters of Amplification Reagentwithout target (negative samples) was added to the other 40 tubes. All80 samples were incubated for 10 min at 62° C., then 5 min at 42° C.Twenty-five microliters of reconstituted Enzyme Reagent was added toeach tube, the rack was removed from the water bath, the rack was shakento mix tube contents, and the rack then was quickly returned to thewater bath. Reaction tube contents were incubated 60 min at 42° C.(amplification), then for 10 min at 80° C. (inactivation of enzymes).Thirty-eight of the positive samples and 38 of the negative samples werepooled and oil was removed from each pool. The two remaining positiveand negative samples were assayed according to the standard Aptima Combo2® manual assay protocol (described above).

B. Preparation of CT+GC rRNA Samples

5×10⁸ copies of CT and GC rRNA prepared by standard procedures wereadded to 100 microliters of UTM:KOVA-Trol™ in a 1:1 ratio (in some cases(indicated in the table below), samples were added to 100 microliters ofSTM). The desired number of replicates of this mixture can be preparedas a pool before spotting on the surface.

C. Deacontamination Assay Protocol

Surface.

Decontamination assays were performed on 2×4 ft sections of ChemSurflaboratory bench (“surface”). Before, between and after the variousexperiments, the surface was cleaned with a 50% bleach solution(household liquid bleach (e.g., Ultra Clorox® Bleach) diluted 1:1 withwater) followed by a water rinse. Wiping was accomplished with papertowels or large Kimwipes.

Sample Application.

One-hundred microliters of each selected sample (see below) was appliedto the surface in a circular spot of about 1.5 inches in diameter.Approximately eight samples were applied, evenly spaced, on the surface.Samples were allowed to dry for approximately 15-30 min.

Sample Collection.

A Gen-Probe endocervical swab was placed in 3 mL of Swab TransportMedium (STM) in a transport tube labeled with the name of the sample tobe collected. The swab was removed from the transport tube and, using acircular motion, each spot was swabbed where the sample was applied.Each swab was returned to its transport tube, the end of the swab wascarefully snapped-off at the scoreline, and the tube was closed usingits penetrable cap, and then vortexed.

Deactivation Formulations Tested.

Among the formulations tested were:

a) 10% bleach one application b) 10% bleach two applications c) 40 mMDCC one application d) 40 mM DCC two applications e) 3% H₂O₂, 1 mMCu(II) one application f) 3% H₂O₂, 1 mM Cu(II) two applications g) 1%H₂O₂, 1 mM Cu(II) one application h) 1% H₂O₂, 1 mM Cu(II) twoapplications i) 200 mM KHSO₅ one application j) 200 mM KHSO₅ twoapplications

Decontamination Protocol.

The decontamination protocol utilized included the following steps:

1. The surface was cleaned (see above).

2. For negative controls a sample was collected from a circular area of˜1.5 inch in diameter, selected randomly on the surface, before anypositive samples were applied to the surface.

3. Approximately eight replicate CT & GC rRNA in UTM:KOVA-Trol™ (1:1)(or STM) samples (100 microliters each) were spotted and evenly spacedon the surface.

4. Spot 1 was treated with decontamination condition “a” above (10%bleach, one application) as follows: the area containing the sample(about 7×7 inch square with sample in the center) was wetted withapproximately 2 mL of reagent (in some cases (indicated in table below)approximately 3 mL was used) and then immediately wiped with a papertowel or large Kimwipe until it was dry (the towel sometimes was flippedover during the process if necessary to complete the drying). The toweland the glove that was on the hand that performed the wiping werecarefully discarded (the other glove was discarded if there was apossibility it became contaminated). A sample from the original spot ofapplication was collected using an endocervical swab as described above.

5. Spot 2 was treated with condition “b” using the same general methoddescribed in “4” above, but also with a second application of thedecontamination reagent.

6. The sample spots then were treated with the decontaminationconditions listed above until all samples on the surface were treated.

7. The surface was cleaned as described above, and a sufficient numberof sample replicates were applied to complete testing of thedecontamination conditions plus one additional spot (to be used as apositive control).

8. Testing of decontamination conditions then was completed.

9. For last remaining sample spot (positive control), the spot wasswabbed directly without any application of decontamination reagent.

10. Steps 1-9 were completed for the negative amplification and thepositive amplification samples.

Assay Protocol.

Replicates (2×400 μL) of each of the samples collected in thedecontamination studies described above were assayed using an AptimaCombo 2® Assay, described below. The assay amplified Chlamydiatrachomatis (referred to herein as “CT” or “Ctr”) and Neisseriagonorrhoeae (referred to herein as “GC” or “Ngo”) template rRNA preparedby standard methodology (“positive Amp”) and also was run withouttemplate rRNA (“negative Amp”). The assay was performed using thefollowing general protocol:

-   -   1. Reconstitute reagents using the docking collars. Reconstitute        Amplification Reagent with Amplification Reconstitution        Solution, Enzyme Reagent with Enzyme Reconstitution Solution,        and Probe Reagent with Probe Reconstitution Solution.    -   2. Dilute Target Capture Reagent (TCR) Component B into Target        Capture Reagent at a 1:100 dilution and mix well by hand.    -   3. Dispense 100 μL of the TCR:Component B mix into each reaction        tube of a Ten-Tube Unit (TTU, Catalog No. TU002; Gen-Probe).    -   4. Pierce the cap and pipette 400 μL of the controls into the        appropriate tube in the following order: Tube 1 (CT Positive        Control) then Tube 2 (GC Positive Control).    -   5. Transfer 400 μL of each sample into the appropriate tube of        the TTU.    -   6. When all samples are loaded in an appropriate rack (Catalog        No. 4579; Gen-Probe), place a sealing card on the TTU, and mix        the samples by gently shaking by hand. Do not vortex the rack.    -   7. Incubate at 62° C. in a water bath for 30 minutes.    -   8. Place the rack on the bench and incubate for 30 minutes.    -   9. Load a Target Capture System (TCS, Catalog No. 5210,        Gen-Probe) with Ten-Tip cassettes (Catalog No. 4578; Gen-Probe).        Ensure that the wash bottle is connected to the pump.    -   10. Prime the pump lines with two flushes of Wash Reagent.    -   11. Place the rack on the TCS magnetic base, remove sealing        cards and cover with new cards (do not stick down). Incubate for        5 minutes.    -   12. Turn on the vacuum for the aspirator. The vacuum gauge must        read between 9 and 11 in. Hg with the system correctly set up.        Aspirate all liquid by lowering the aspiration manifold slowly        into the bottom of the tubes. Tap the bottom of the tubes with        the tips briefly. Avoid holding the tips at the bottom of the        tube. Aspirate until the all foam is removed from the tube.    -   13. Add 1.0 mL of Wash Reagent into each tube, by pumping the        wash bottle once.    -   14. Cover tubes with a sealing card and vortex on the        multi-vortexer.    -   15. Place rack on the TCS magnetic base for 5 minutes.    -   16. Aspirate all liquid.    -   17. Add 75 μL of the reconstituted Amplification Reagent.    -   18. Add 200 μL of Oil Reagent.    -   19. Cover tubes with a sealing card and vortex on the        multi-vortexer.    -   20. Incubate the rack in a 62° C. water bath for 10 minutes.    -   21. Transfer the rack to a circulating water bath at 42° C. and        incubate for 5 minutes.    -   22. With the rack in the water bath, remove the sealing card,        and add 25 μL of the Enzyme Reagent to all of the reactions.    -   23. Immediately cover with a sealing card, briefly remove from        the waterbath, and mix the reactions, gently shaking by hand.    -   24. Incubate the rack at 42° C. for 60 minutes.    -   25. Remove the rack from the water bath and transfer to the HPA        area. Add 100 μL of the reconstituted Probe Reagent.    -   26. Vortex on the multi-vortexer.    -   27. Incubate the rack in a circulating water bath at 62° C. for        20 minutes.    -   28. Remove the rack from the water bath and incubate on the        bench-top, at room temperature, for 5 minutes.    -   29. Add 250 μL of Selection Reagent.    -   30. Cover tubes with a sealing card and vortex on the        multi-vortexer.    -   31. Incubate the rack at 62° C. in a circulating water bath for        10 minutes.    -   32. Incubate the rack on the bench-top, at room temperature, for        15 minutes.    -   33. Light-off the reactions in a LEADER® HC+ Luminometer        (Catalog No. 4747; Gen-Probe) Combo software.

Before assay, Ngo/Ctr rRNA samples were prepared by spikingamplification-negative samples with 0.5 fg of CT rRNA (about 2×10²copies) and 50 fg of GC rRNA (about 2×10⁴ copies). In addition, 5-10negative assay controls (STM only) were performed. Acceptance criteriawere as follows:

Controls Specifications Amplification Positive Control, CT CT Positive,GC Negative Amplification Positive Control, GC CT Negative, GC PositiveSamples Specifications Negative control CT Negative, GC Negative (swipesfrom clean, control area) Positive control CT Positive, GC Positive(swipes from sample spot w/ no cleaning) rRNA and positive ampliconswipes CT Negative, GC Negative (cleaned areas) Negative amplicon(cleaned areas) CT Positive, GC Positive

Follow-Up Testing.

Any samples not meeting the above specifications were stored at roomtemperature and re-tested the following day. The acceptance criteria forthe follow-up testing are the same as the acceptance criteria for theinitial testing (see above).

D. Characterization Results of Nucleic Acid Deactivation Using VariousFormulations and Application Methods

Table 12 below depicts results collected using the protocols describedabove. “NA Source” is the nucleic acid source, “# App” is the number ofreagent applications, “kRLU” is relative light units times a factor of1000, and “pip” is piperazine. Expected Ctr and Ngo results are negative(Neg) for Ngo/Ctr rRNA, Neg for Pos Amplification and positive (Pos) forNeg Amp. The majority of Ctr and Ngo results from the tests were valid,and invalid results are not included in the table.

TABLE 12 Effectiveness of Reagents Used for Surface Decontamination CtrNgo Reagent NA Source # App. kRLU Result Result 10% Bleach Ngo/Ctr rRNA1 10 Neg Neg 1 6 2 5 1 3 1 3 1 3 2 2 2 2 Positive 1 3 Amplification 1 3(100 μL) 2 3 2 3 1 2 1 2 2 3 2 2 Negative 1 828 Pos Pos Amplification 1859 (100 μL) 2 825 2 870 1 1004 1 1020 2 996 2 1008 10% Bleach, 0.1MNgo/Ctr rRNA 1 9 Neg Neg Bicarb, 0.025% LLS 1 9 Neg Neg 2 10 Neg Neg 210 Neg Neg 1 10 Neg Neg 1 10 Neg Neg 1 12 Neg Neg 1 11 Neg Neg 2 10 NegNeg 2 11 Neg Neg Positive 1 10 Neg Neg Amplification 1 11 Neg Neg (100μL) 2 11 Neg Neg 2 11 Neg Neg 1 8 Neg Neg 1 8 Neg Neg 2 10 Neg Neg 2 12Neg Neg 1 9 Neg Neg 1 7 Neg Neg 2 11 Neg Neg 2 11 Neg Neg Negative 12243 Pos Pos Amplification 1 2269 Pos Pos (100 μL) 2 2240 Pos Pos 2 2259Pos Pos 1 2213 Pos Pos 1 2348 Pos Pos 2 2353 Pos Pos 2 2277 Pos Pos 10%Bleach, 0.1M PB, Ngo/Ctr rRNA 1 10 Neg Neg 0.05% SDS 1 11 Neg Neg 2 10Neg Neg 2 11 Neg Neg 1 8 Neg Neg 1 8 Neg Neg 1 11 Neg Neg 1 10 Neg Neg 29 Neg Neg 2 8 Neg Neg Positive 1 11 Neg Neg Amplification 1 13 Neg Neg(100 μL) 2 7 Neg Neg 2 8 Neg Neg 1 12 Neg Neg 1 11 Neg Neg 1 11 Neg Neg1 11 Neg Neg Negative 1 2192 Pos Pos Amplification 1 2285 Pos Pos (100μL) 2 2240 Pos Pos 2 2212 Pos Pos 1 806 Neg Pos 1 899 Pos Pos 2 2218 PosPos 2 2193 Pos Pos 40 mM DCC Ngo/Ctr rRNA 1 3 Neg Neg 1 3 Neg Neg 2 3Neg Neg 2 3 Neg Neg 1 3 Neg Neg 1 2 Neg Neg 2 3 Neg Neg 2 3 Neg Neg 1 12Neg Neg Positive 1 3 Neg Neg Amplification 1 3 Neg Neg (100 μL) 2 3 NegNeg 2 3 Neg Neg 1 15 Neg Neg 2 2 Neg Neg 2 2 Neg Neg 1 7 Neg Neg 1 8 NegNeg Negative 1 812 Pos Pos Amplification 1 795 Pos Pos (100 μL) 2 726Pos Pos 2 668 Pos Pos 1 886 Pos Pos 1 919 Pos Pos 2 937 Pos Pos 2 919Pos Pos 3% peroxide, 1 mM Ngo/Ctr rRNA 1 6 Neg Neg CuSO₄ 1 3 Neg Neg 2 3Neg Neg 2 3 Neg Neg 1 3 Neg Neg 1 3 Neg Neg 2 8 Neg Neg 2 3 Neg NegPositive 2 11 Neg Neg Amplification (10 μL) Negative 2 870 Pos PosAmplification 2 865 Pos Pos (100 μL) Negative 2 781 Pos PosAmplification 2 784 Pos Pos (100 μL) 3 mL 3% peroxide, Positive 2 7 NegNeg 1 mM CuSO₄ Amplification 2 6 Neg Neg (10 μL) 3% peroxide, 1 mMNgo/Ctr rRNA 2 8 Neg Neg CuSO₄, 25 mM pip 1 21 Neg Neg 1 8 Neg Neg 2 8Neg Neg 2 9 Neg Neg 3% peroxide, 1 mM Ngo/Ctr rRNA 2 8 Neg Neg CuSO₄, 50mM pip 2 22 Neg Neg 1 158 Neg Neg 2 10 Neg Neg 2 12 Neg Neg 2 10 Neg Neg2 3 Neg Neg Ngo/Ctr rRNA 2 10 Neg Neg in STM 2 11 Neg Neg 3% peroxide, 2mM Ngo/Ctr rRNA 2 7 Neg Neg CuSO₄, Ngo/Ctr rRNA 2 11 Neg Neg 50 mM HEPESin STM 2 10 Neg Neg 3% peroxide, 2 mM Ngo/Ctr rRNA 1 9 Neg Neg CuSO₄, 210 Neg Neg 50 mM pip 2 9 Neg Neg (10 day Cu/pip) 1 7 Neg Neg 2 8 Neg Neg2 8 Neg Neg 3% peroxide, 2 mM Ngo/Ctr rRNA 2 11 Neg Neg cupric acetate 212 Neg Neg 2 9 Neg Neg 2 9 Neg Neg 2 10 Neg Neg 2 10 Neg Neg Ngo/CtrrRNA 2 11 Neg Neg in STM 2 10 Neg Neg 3% peroxide, 2 mM Ngo/Ctr rRNA 2 9Neg Neg CuSO₄ 2 9 Neg Neg 2 10 Neg Neg 2 9 Neg Neg Ngo/Ctr rRNA 2 10 NegNeg in STM 2 9 Neg Neg 1% peroxide, 1 mM Ngo/Ctr rRNA 1 3 Neg Neg CuSO₄1 3 Neg Neg 2 3 Neg Neg 2 3 Neg Neg 2 3 Neg Neg 2 2 Neg Neg 1 7 Neg NegPositive 2 7 Neg Neg Amplification 2 7 Neg Neg (100 μL) 200 mM KHSO₅Ngo/Ctr rRNA 1 3 Neg Neg 1 3 Neg Neg 2 3 Neg Neg 2 3 Neg NegThe results in the table show bleach-containing reagents—including thosethat also contain a corrosion inhibitor and a surfactant—effectivelydeactivated rRNA and positive and negative TMA reactions on surfaces.The same was true for solutions containing 40 mM DCC. Solutionscontaining peroxide and copper effectively deactivated rRNA on surfaces,and were not as efficacious as bleach for consistently decontaminatingsurfaces of positive or negative TMA reactions under the conditionstested. Adding piperazine or HEPES to the peroxide/copper solutions didnot significantly alter deactivation performance on surfaces under theconditions tested. Peroxymonosulfate deactivated rRNA on surfaces, butnot positive and negative TMA reactions under the conditions tested.

Example 13 Characterization of Nucleic Acid Deactivation Formulations

Effects of including corrosion inhibitors, surfactants and fragrances innucleic acid deactivation formulations were assessed. Bleach, and to alesser but still significant extent DCC, cause corrosion of metals andother materials. Nucleic acid deactivation activity of various candidateanti-corrosion compounds, including the sodium salts of phosphate (PB),borate, bicarbonate and dodecyl sulfate (SDS), were tested in solutionprior to analysis using real-time TMA and PAGE (e.g., Example 8 andExample 1). Studies were performed to test the activity of bleach andDCC when mixed together with the candidate corrosion inhibitors.Phosphate at pH 6.4 and 7.5 destabilized bleach (loss of activityincreased with time) whereas phosphate at pH 9.1 or 9.5 did not. Theconverse was true for DCC, where the higher pH phosphate's (9.1 and 9.5)were destabilizing whereas the lower pH phosphate's (6.4 and 7.5) werenot. Bleach was stable in borate at pH 7.6 or 9.1 and bicarbonate at pH9.3. SDS did not have any apparent effect on the activity of bleach.

Anti-corrosion formulations with bleach were also tested with thesurface decontamination protocol described in Example 12. Allformulations tested were determined to be effective, thus demonstratingthe anti-corrosion agents have no apparent negative effect on bleachactivity. One application (“1 app”) is one application of the reagentand two applications (“2 app”) is two applications of the reagent.Results from the analysis are presented in Table 13 below.

TABLE 13 Anti-Corrosion Formulations Reagent Contamination Source Result10% Bleach, 100 mM Bicarb., rRNA(Ctr/Ngo) Validated 0.025% LLS, 1 App.10% Bleach, 100 mM Bicarb., rRNA(Ctr/Ngo) Validated 0.025% LLS, 2 App.10% Bleach, 100 mM Bicarb., Pos. Amplicon (100 μL) Validated 0.025% LLS,1 App. 10% Bleach, 100 mM Bicarb., Pos. Amplicon (100 μL) Validated0.025% LLS, 2 App. 10% Bleach, 100 mM Bicarb., Neg. Amplicon (100 μL)Validated 0.025% LLS, 1 App. 10% Bleach, 100 mM Bicarb., Neg. Amplicon(100 μL) Validated 0.025% LLS, 2 App. 10% Bleach, 100 mM PB,rRNA(Ctr/Ngo) Validated 0.05% SDS, 1 App. 10% Bleach, 100 mM PB,rRNA(Ctr/Ngo) Validated 0.05% SDS, 2 App. 10% Bleach, 100 mM PB, Pos.Amplicon (100 μL) Validated 0.05% SDS, 1 App. 10% Bleach, 100 mM PB,Pos. Amplicon (100 μL) Validated 0.05% SDS, 2 App. 10% Bleach, 100 mMPB, Neg. Amplicon (100 μL) Validated 0.05% SDS, 1 App. 10% Bleach, 100mM PB, Neg. Amplicon (100 μL) Validated 0.05% SDS, 2 App.

An assay for assessing corrosion was devised. The assay comprisedsoaking stainless steel bolts (1″ long, ⅛″ diameter, standard thread,hex-head stainless steel bolts) in candidate solutions and visuallyscoring corrosion over time. Results from the corrosion inhibitionstudies are summarized in Table 14 below.

TABLE 14 Corrosion Inhibition Result Agent Corrosion InhibitionPhosphate, pH 9.1 High Phosphate, pH 9.5 High Borate, pH 7.6 ModerateBorate, pH 8.5 Moderate Bicarbonate, pH 9.3 High SDS* Low to moderateSDS + other corrosion inhibitors SDS enhanced activity of corrosioninhibitor *Other detergents/surfactants (including lithium laurylsulfate, Photo-Flo ®, saponin, Triton ® X-100 and General UseHybridization Reagent (Gen-Probe)) were tested, with similar results asfor SDS.

Detergents and surfactants also were tested for effects on the physicalproperties of bleach solutions on surfaces. These agents decreasedsurface tension and allowed for more complete wetting of the surfacewith the bleach solution (typically 0.6% hypochlorite). To decreasefoaming of the solution when applied to the surface, detergentconcentration was lowered to a level that minimized foaming but retainedeffective surfactant qualities. SDS and LLS levels of approximately0.005% to 0.02% (w/v) minimized foaming in this particular application.

Effects of fragrances on activity and stability of bleach and DCC alsowere tested. Among the fragrances tested were 2141-BG, 2145-BG, and twoother custom fragrances from International Flavors and Fragrance. Thefragrances exhibited no detectable effect on activity and stability of10% bleach and DCC according to PAGE analysis. Also, the fragrancesexhibited no detectable effect on corrosion inhibition of variouscompounds tested (e.g., phosphate and bicarbonate).

As a culmination of results for corrosion inhibitors,detergent/surfactants and fragrances, formulations of these reagentswith bleach were developed. Unexpectedly, the balance between componentswas critical for maintaining physical stability of the solution. Therewere various combinations of these components that were successful inthis regard. One formulation was as follows:

corrosion inhibitor/detergent/fragrance (6.7× concentrate): 600 mMbicarbonate (pH 9.3), 0.1% SDS, 0.05% 2141-BG

finished decontamination reagent: 0.6% hypochlorite, 90 mM bicarbonate(pH 9.3), 0.015% SDS, 0.0075% 2141-BG.

Solutions comprising peroxide and copper were further characterized. Itwas discovered that UTM stimulated inactivation of rRNA in solutionscontaining peroxide and Cu(II). The effects of the individual componentsof the UTM formulation (150 mM HEPES, pH 7.6, 300 mM LLS, 10 mM(NH₄)₂SO₄) were examined, and it was discovered that the HEPES wasresponsible for the stimulation. Effects of pH and concentration on theobserved inactivation of rRNA then was examined. The activity ofdifferent chemical components of HEPES (ethanol, ethanesulfonic acid andpiperazine) and PIPES, a buffer similar to HEPES, also were examined. Itwas discovered piperazine was essentially as active as HEPES, andpiperazine at a pH of 5.5 was utilized for further characterization. Italso was discovered that piperazine stabilized Cu(II) in solution in achemical configuration that maintains activity with peroxide forinactivating nucleic acids.

Example 14 Stability of Nucleic Acid Deactivation Formulations

Selected reagents were stored under a variety of conditions. At selectedtime points, the formulations were assayed for the ability to deactivatetarget nucleic acid using a solution assay, in which rRNA was incubatedwith reagents in solution, diluted, and an aliquot was assayed usingreal-time TMA (Example 8). Incubation conditions were at roomtemperature with no protection from light. Results are providedhereafter.

I. 40 mM CuSO₄/1 M Piperazine (Acetate), pH 5.5

Incubation Solution Stability Time (Days) Characteristics (% Day 0) 0Clear, royal blue 100 1 100 7 100 13 100 41 100 63 96 70 94 139 94 185Getting lighter 85

II. 80 mM CuSO₄/1M Piperazine (Acetate), pH 5.5

Incubation Solution Stability Time (days) Characteristics (% Day 0) 0Clear, royal blue 100 83 100 129 100

III. 200 mM CuSO₄ (in Water)

A. Stored at Room Temperature, No Protection from Light

Incubation Solution Stability Time (Days) Characteristics (% Day 0) 0Clear, pale blue 100 4 97 50 50

IV. 10% Bleach/Sodium Bicarbonate/SDS/IFF

A. 10% Bleach/0.2 M Sodium Bicarbonate (pH 9.3)/0.05% SDS

Incubation Solution Stability Time (days) Characteristics (% Day 0) 0Clear 100 1 100 4 100 34 100 57 100 72 100

B. 10% Bleach/0.08M Sodium Bicarbonate (pH 9.3)/0.020% SDS/0.025%2141-BG

Incubation Solution Stability time (Days) Characteristics (% Day 0) 0Clear, pale 100 15 yellow 100 20 100 26 100

C. 10% Bleach/0.09M Sodium Bicarbonate (pH 9.3)/0.015% SDS/0.0075%2141-BG

Incubation Solution Stability Time (days) Characteristics (% Day 0) 0Clear 100 7 100 28 100 58 96 103 100 148 100 214 40 242 35

V. Sodium Bicarbonate/SDS/2141-BG (then Added to “Fresh” Bleach)

A. 600 mM Sodium Bicarbonate (pH 9.3)/0.1% SDS/0.05% 2141-BG

-   -   (6.7× solution)

Incubation Stability Time (days) Solution Characteristics (% Day 0) 0Clear, pale yellow; fine 100 2 white particulate on 100 bottom of tube31 Increased particulates 100 58 Same as day 31 100 103 100 148 100 214100 242 100 276 100 330 100

B. 600 mM Sodium Bicarbonate (pH 9.3)/0.1% SDS/0.05% 2145-BG

-   -   (6.7× solution)

Incubation Solution Stability Time (days) Characteristics (% Day 0) 0Clear 100 24 100 69 100 126 100 154 100 188 100 242 100

Example 15 Deactivation of Nucleic Acid on Laboratory Equipment

Formulations and procedures for deactivating nucleic acid on severalpieces of laboratory equipment, including a vacuum trap system, anaspiration manifold, a rack and a deck, were assessed for efficacy.

A. Vacuum Trap System

A vacuum system comprising an aspiration manifold, two traps, an inlinefilter, and a vacuum pump connected in series by tubing was utilized forconducting an amplification assay after multiple target capture runs(both Ctr and Ngo rRNA). Contamination was assessed without addingbleach to the first trap. After the runs, swab samples were taken fromvarious locations in the vacuum system and assayed for presence of Ctrand Ngo rRNA using the real-time TMA assay presented in Example 8. Nodetectable contamination with Ngo rRNA was identified outside of thefirst trap. Contamination with Ctr rRNA was identified in the tubingbetween the first and second traps, in the second trap and in the tubingbetween the second trap and the inline filter, and no contamination wasdetected after the inline filter. These results demonstrated that nodetectable Ngo or Ctr rRNA escaped into the environment, and it istherefore feasible to not to include bleach in the first trap duringusage.

B. Aspiration Manifold

One protocol for decontaminating a target capture aspiration manifoldutilized for a TMA assay (Aptima Combo 2® Assay) included the step ofsoaking the manifold in 50% bleach for 10 minutes followed by thoroughrinsing with water. This procedure resulted in corrosion of the manifoldand the relatively frequent need to replace it.

To test other decontamination protocols and agents, the manifold wasintentionally contaminated, decontamination was attempted, thencontamination levels measured. Each of target negative samples (10replicates) remained negative using the contaminated manifold,demonstrating that the target capture system prevented contaminationfrom entering new samples. In one decontamination protocol, it wasdiscovered that leaving the manifold attached to the system andaspirating nucleic acid deactivation formulations through itsuccessfully decontaminated the manifold. In such a procedure, it wasdetermined 0.6% hypochlorite (10% bleach) or 40 mM DCC (followed by awater rinse) successfully decontaminated the manifold. A hydrogenperoxide/copper solution also successfully decontaminated the manifold,but this reagent was not as suitable for routine use as it couldvigorously evolve oxygen when under reduced pressure in the vacuumsystem. It was determined that aspirating approximately 50 mL (about 5mL per nozzle) of a 0.6% hypochlorite solution (with corrosioninhibitor, detergent and fragrance) followed by approximately 50 mL(about 5 mL per nozzle) of water, and then leaving the vacuum pump onfor at least 1 minute sufficiently decontaminated the aspirationmanifold.

C. Tecan Deck Decontamination

Leading bleach alternative candidates were tested for decontamination ofthe deck of the DTS® Tecan Genesis System (Catalog No. 5216 or 5203;Gen-Probe). The results in Table 15 below were observed.

TABLE 15 Deck Decontamination Reagent Degree of Effectiveness 10% bleach100% 40 mM DCC 100% 3% peroxide, 1 mM Cupric Sulfate 100%Thus, multiple formulations and procedures effectively deactivatednucleic acids that contaminated various laboratory equipment.

Example 16 Efficacy of Nucleic Acid Deactivation Formulation and Methodsat Two Laboratory Sites

Efficacy of two decontamination reagents and methods in a clinicallaboratory setting were characterized at two sites. Reagent 1 (3% H₂O₂(w/v), 2 mM cupric sulfate) and Reagent 2 (0.6% hypochlorite (w/v), 90mM bicarbonate, 0.015% SDS (w/v), 0.0075% (v/v) 2141-BG), used accordingto the prescribed protocol provided to each site (see below), wereequivalent to the protocol using 50% bleach described in the packageinsert for the Aptima Combo 2® Assay kit (Catalog No. 1032) and at httpaddress www.gen-probe.com/pdfs/pi/IN0037-04RevA.pdf, and yieldedeffective nucleic acid deactivation and decontamination control fornucleic acid assay procedures in a clinical laboratory setting.

A. Materials

Following is a list of materials utilized at each site:

-   -   Reagent 1A (3% H₂O₂ USP grade)    -   Reagent 1B (copper sulfate, dry powder)    -   Reagent 2A (600 mM sodium bicarbonate, 0.1% (w/v) sodium        dodecylsulfate, 0.05% 2415-BG fragrance)    -   Household bleach (˜6% hypochlorite)    -   De-ionized (or higher quality) water    -   Milli-Q (or equivalent quality) water    -   Aptima Combo 2® Test Kit    -   Dual Positive Control (CT and GC rRNA)    -   Negative Control    -   Squirt bottle with a vented top (for Reagent 1)

B. Procedures

The following procedures were utilized at each site. For each rack ofsamples (up to 10 Ten-Tube Units (TTUs; Catalog No. TU0022; Gen-Probe)run in the Aptima Combo 2® Assay, included were the usual two-runcontrols (Positive Control, CT and Positive Control, GC), two DualPositive Controls (see Materials), 16 Negative Controls (see Materials)and up to 80 patient specimens. The assay was performed according to thestandard protocol (package insert).

If the two-run controls met run control criteria, the run was valid(PASS). If one or both of the run controls did not meet run controlcriteria, the run was invalid (FAIL) and all results in the same runwere invalid and were not reported. The run was then repeated. Also, asusual for patient samples, initial equivocal or invalid results wererepeated.

Described below are the three phases of the research study. Each stagewas run between 2 and 4 weeks as less than 2 weeks might not allowadequate evaluation of the decontamination protocol. Three weeks wasdetermined as being ideal, and the maximum duration was four weeks. Theentire study was expected to be completed in 9 weeks, with a maximumduration of 12 weeks. For each phase of the study, 15 racks of sampleswere assayed, with all containing the appropriate controls as describedabove.

Phase 1: The standard Aptima Combo 2® protocol utilizing 50% bleach wasused for decontamination as described in the package insert (httpaddress www.gen-probe.com/pdfs/pi/IN0037-04RevA.pdf). This approach wasutilized to establish a baseline of results for comparison with resultsobtained when the test decontamination protocol was used.

Phase 2: The test decontamination protocol was utilized (see below).

Phase 3: The test decontamination protocol (see below) was utilized,except Reagent 2 was used when the protocol called for use of Reagent 1.Reagent 2 still was utilized when the protocol called for use of Reagent2.

1. Rack Set-Up

Each laboratory was instructed to utilize the following procedure forsetting-up racks of samples:

-   -   1. Begin rack set-up in the standard fashion as described in the        package insert.    -   2. Add 400 μL of the Positive Control, CT, to reaction tube 1.    -   3. Add 400 μL of the Positive Control, GC, to reaction tube 2.    -   4. Add 400 μL of the Dual Positive Control to reaction tubes        3-4.    -   5. Add 400 μL of the Negative Control to reaction tubes 5-20.    -   6. Add 400 μL of patient specimens into reaction tubes 21 up to        100.

2. General Decontamination Protocol

Each laboratory was instructed to apply good physical containmenttechniques in order to guard against spread of contamination in the labwhile decontaminating each workspace. Each laboratory was cautioned thatthe glove on the hand used for cleaning would become contaminated andthat touching clean objects with this hand should be avoided. It wasrecommended that one hand should be reserved for cleaning only and theother hand (clean) for application of reagent only. It also wasrecommended that used towels and gloves should be discarded in areceptacle in which they would be well-contained, making sure that nodripping occurred between the area undergoing decontamination and thereceptacle.

3. Reagent Preparation

Each laboratory was instructed to prepare the following reagents usingthe procedures outlined below:

-   -   a. Prepare Reagent 1B (every 2 weeks)        -   i. Add 30 mL of Milli-Q (or equivalent quality) water to 1            vial of Reagent 1B (dry reagent).        -   ii. Tightly cap and invert 30 times. Let stand for 1 minute.            Invert 30 more times. Make sure all of the dry reagent is            dissolved.        -   iii. Between uses (see section 2b below), store Reagent 1B            (liquid) at 2-8° C. in the dark (the dry reagent can be            stored at room temperature).        -   iv. After 2 weeks of storage, discard Reagent 1B (liquid)            and prepare a fresh solution.    -   b. Prepare Reagent 1 (daily)        -   i. Add 150 mL of Reagent 1A to a squirt bottle with a vented            top (provided).        -   ii. Add 1.5 mL of Reagent 1B to the squirt bottle.        -   iii. Replace top and thoroughly mix by swirling contents for            10-15 seconds        -   iv. Use contents as described below. If there is any escape            of Reagent 1 from the squirt bottle between uses, loosen the            top and then retighten immediately before resuming use.        -   v. After the last use of the day or 12 hours, whichever            comes first, dispose of any remaining Reagent 1 in the            squirt bottle. Prepare fresh reagent as described above when            needed.    -   c) Prepare Reagent 2 (every 2 weeks)

The recipe provided below is for the preparation of 1 liter of Reagent2. The actual amount made is to be determined based on the anticipatedreagent usage in a given laboratory. The preparation of Reagent 2 to beused for cleaning racks and other equipment and may be performed in thevessel used for soaking.

-   -   i. Add 750 mL of de-ionized (or higher quality) water to an        appropriate vessel. Add 150 mL of Reagent 2A to the vessel,        followed by 100 mL of household bleach (this step can be        performed in a fume hood if so desired to avoid contact with        bleach fumes).    -   ii. Close container and thoroughly mix by swirling contents for        15-20 seconds.    -   iii. Use contents as needed.    -   iv. At the end of 2 weeks, discard any unused Reagent 2 and        prepare a fresh solution as described above.

4. Pre-Assay Procedures

Each laboratory was instructed to perform the following pre-assayprocedures.

-   -   a. Turn on the water baths in the pre-amp area, but not the        post-amp area (if the water baths are routinely left on 24 hours        a day, this practice can be continued; however, the person        running the Aptima Combo 2® Assay in a given day should not        enter the post-amp area until the assay is ready to proceed in        that area (see below)).    -   b. Clean all surfaces in the pre-amp area as follows (in the        order listed):        -   Tecan. Using a squirt bottle, wet a paper towel with Reagent            1 until the towel is saturated but not dripping. Thoroughly            wet and clean the Tecan deck with the wet towel (do not            include a 1 minute incubation time as in the current            standard protocol) and continue wiping until all the            surfaces are dry. This may require additional wetted towels            as well as dry towels. Once the surface has been cleaned and            dried, repeat this procedure with a second application of            Reagent 1. Do not rinse with water.        -   TCS Unit. Using a squirt bottle, wet a paper towel with            Reagent 1 until the towel is saturated but not dripping.            Thoroughly wet and clean surfaces of the TCS (Catalog No.            5202; Gen-Probe) with the wet towel (do not include a 1            minute incubation time as in the current standard protocol)            and continue wiping until all the surfaces are dry. This may            require additional wetted towels as well as dry towels. Once            the surface has been cleaned and dried, repeat this            procedure with a second application of Reagent 1. Do not            rinse with water.        -   Bench surfaces. Liberally apply Reagent 1 to the bench            surface using a squirt bottle Immediately clean the surface            using a paper towel, making certain that the entire surface            has been thoroughly wetted with the decontamination reagent            yet taking care to not splash the reagent onto the floor,            into surrounding areas, etc. Do not include a 1 minute            incubation time as in the current standard protocol.            Continue wiping until the entire surface is dry. This may            require more than one paper towel. Repeat this procedure            with a second application of Reagent 1. Do not rinse with            water.        -   Pipettors. Using a squirt bottle, wet a paper towel with            Reagent 1 until the towel is saturated but not dripping.            Thoroughly clean the surfaces of the pipet with the wet            towel (do not include a 1 minute incubation time as in the            current standard protocol) and continue wiping until the            pipet is dry. Repeat this procedure with a second            application of Reagent 1. Do not rinse with water.    -   c. When finished cleaning the pre-amp area, carefully discard        both gloves. Change gloves sooner if there is any suspicion of        possible cross contamination.

5. Post-Specimen Preparation Procedures

Each laboratory was instructed to perform the following post-specimenpreparation procedures:

-   -   a. Carefully discard gloves used during specimen preparation and        replace with clean gloves.    -   b. Clean the Tecan, items to be soaked (see below), bench        surfaces used in specimen processing area and any pipettors used        as follows:        -   i. Tecan. Clean with Reagent 1 as described above and            carefully discard both gloves.        -   ii. Items to be soaked. After use, completely submerge            racks, reagent reservoirs, deck plates, disposable tip racks            and waste chute (and any other items that you currently            soak) in Reagent 2 and allow to soak for 30-60 minutes.            Rinse thoroughly with running water (do not soak in a bath            of rinse water) and then dry completely with paper towels            (air drying is acceptable). Carefully discard both gloves.        -   iii. Bench surfaces. Clean with Reagent 1 as described            above. Carefully discard both gloves        -   iv. Pipettors. Clean with Reagent 1 as described above.            Carefully discard both gloves

6. Post-Target Capture Procedures

Each laboratory was instructed to employ the following post-targetcapture procedures:

-   -   a. Aspiration manifold. Place a new Ten-Tip Cassette (TTC;        Catalog No. 4578; Gen-Probe) into the TCS. Turn on the vacuum        pump. Carefully attach the manifold to the tips in the TTC.        Carefully aspirate all Wash Solution remaining from the Aptima        Combo 2® Assay run from the priming trough of the Wash Solution        dispense station (the Wash Solution dispense manifold will have        to first be moved out of the way). Add 100 mL of Reagent 2 to        the trough, then carefully aspirate it through the aspiration        manifold. Add 100 mL of de-ionized water to the trough, then        carefully aspirate it through the aspiration manifold. Eject the        tips into their original TTC. Leave the vacuum pump on for at        least 1 minute after the last aspiration.    -   b. TCS, bench surfaces and pipettors. Clean with Reagent 1 as        described above. Carefully discard both gloves    -   c. Vacuum trap waste liquid. When required (see below),        decontaminate the liquid in the Waste Bottle. Attach the Waste        Bottle to the TCS unit empty (i.e., do not add bleach). Use the        Waste Bottle until it is 25% full (i.e., available capacity not        to exceed 25%) or for 1 week (whichever is first). Remove the        Waste Bottle from the system and carefully add 400 mL of        undiluted bleach (if desired, this procedure can be performed in        a fume hood in order to avoid release of fumes into the        laboratory). Cap the Waste Bottle and gently swirl the contents        until fully mixed. Incubate 5 minutes, then pour the waste into        a sink. Reconnect the empty Waste Bottle to the TCS unit. Use        universal precautions when handling and disposing of liquid and        solid waste. Dispose of liquid and solid waste according to        local, state, and federal regulations. The contents of the Waste        Bottle should be treated as a potential source of assay        contamination. Take precautions to avoid contaminating the work        surfaces. Carefully discard both gloves.

7. Amplification Reaction b Procedures

Each laboratory was instructed to perform the following procedures aftereach amplification reaction was started, which is the last stepperformed in the pre-amp area. After starting the reaction, eachlaboratory was instructed to clean the bench tops surrounding the waterbaths, the handles to the lids of the water baths and the pipettorsusing Reagent 1 according to the procedures described above. Eachlaboratory was instructed to carefully discard both gloves afterperforming these procedures.

8. Post-Amp Area Procedures

Each laboratory was provided with the following instructions concerningpost-amplification area procedures. After the last cleaning in thepre-amp area was completed and new gloves were adorned, each laboratorywas instructed to immediately turn on the 62° C. water bath afterentering the pre-amp area. Instructions also were to pre-clean allsurfaces in the post-amp area (lab benches, pipettors, handles, andothers) using Reagent 2 according to the specific procedures describedabove, and then to carefully discard both gloves.

9. Post Amplification Procedures

Each laboratory was provided with the following instructions concerningpost-amplification procedures. After adorning a clean set of gloves,instructions were provided to carefully remove the rack(s) from the 42°C. water bath, and to avoid contaminating the lid of the water bath.

10. Post Detection Procedures

Each laboratory was provided with the following instructions concerningpost-detection procedures. Instructions were to (a) remove TTU's fromthe luminometer and deactivate reactions using the current procedure inthe product insert; (b) clean all surfaces (bench surfaces, pipettors,handle on water bath lid, exterior of the LEADER® HC+Luminometer, andothers) using Reagent 2 according to the specific procedures describedabove, (c) every two weeks, or as needed, clean the interior of the HC+with DI water as currently described in the operator's manual and soakthe HC+ cassettes in Reagent 2 for 30-60 minutes, and (d) carefullydiscard both gloves.

11. Acceptance Criteria

Each laboratory was instructed to use the following acceptance criteria.

Controls Specifications Amplification Positive Control, CT CT Positive,GC Negative Amplification Positive Control, GC CT Negative, GC PositiveNegative Controls CT Negative, GC Negative Dual Positive Controls CTPositive, GC Positive

C. Results

Reagents 1 and 2 used according to the prescribed protocol wereequivalent to the protocol using 50% bleach provided with the AptimaCombo 2® Assay kit, and yielded effective decontamination control forthe Aptima Combo 2® Assay in a clinical setting (see Table 16 below).

TABLE 16 Analysis of Negative and Positive Control Data Fisher's PhaseExact P Laboratory Sample Result I II III Total Value Laboratory INegative Equivocal 1 0 2 3 0.625 Control Low 1 0 2 3 Positive Negative238 240 252 730 Total 240 240 256 736 Positive High 30 30 32 92 ControlPositive Negative 0 0 0 0 Total 30 30 32 92 Laboratory II NegativeEquivocal 1 0 0 1 0.110 Control High 1 0 0 1 Positive Low 1 0 0 1Positive Negative 237 240 240 717 Total 240 240 240 720 Positive High 3030 30 90 Control Positive Negative 0 0 0 0 Total 30 30 30 90When the new decontamination reagents and protocol were used, 540 of 540(100%) control samples for Phase II and 554 of 558 (99.3%) controlsamples for Phase III yielded the expected results. When 50% bleach withthe standard protocol was used (Phase I), 535 of 540 (99.1%) controlsamples yielded the expected results. A Fisher's exact test (astatistical hypothesis test method to demonstrate statisticaldifferences between multiple groups with qualitative outcomes;Categorical Data Analysis by Alan Agresti (1990), pages 59-67, 68, 70,78, 488, John Wiley & Sons, New York, N.Y.) was performed on the datausing SAS Version 8.2 software. It is widely accepted that P<0.05suggests a significant difference between groups while P>0.05 isindicative of no statistical difference. The Fisher's exact test yieldeda p value of 0.625 for assays run at Laboratory 1 and 0.110 for assaysrun at Laboratory II. These results indicate statistical equivalencebetween the conditions of all three phases.

The entirety of each patent, patent application, publication anddocument referenced herein hereby is incorporated by reference. Citationof the above patents, patent applications, publications and documents isnot an admission that any of the foregoing is pertinent prior art, nordoes it constitute any admission as to the contents or date of thesepublications or documents. Incorporation by reference of thesedocuments, standing alone, should not be construed as an assertion oradmission that any portion of the contents of any document is consideredto be essential material for satisfying any national or regionalstatutory disclosure requirement for patent applications.Notwithstanding, the right is reserved for relying upon any of suchdocuments, where appropriate, for providing material deemed essential tothe claimed subject matter by an examining authority or court.

Modifications may be made to the foregoing without departing from thebasic aspects of the disclosure. Although the disclosure has beendescribed in substantial detail with reference to one or more specificembodiments, those of ordinary skill in the art will recognize thatchanges may be made to the embodiments specifically disclosed in thisapplication, and yet these modifications and improvements are within thescope and spirit of the disclosure. The disclosure illustrativelydescribed herein suitably may be practiced in the absence of anyelement(s) not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. Thus, the terms and expressions which have been employed are usedas terms of description and not of limitation, equivalents of thefeatures shown and described, or portions thereof, are not excluded, andit is recognized that various modifications are possible within thescope of the disclosure. Embodiments of the disclosure are set forth inthe following claims.

1. A composition for deactivating nucleic acids, the compositioncomprising: hydrogen peroxide at a concentration of about 0.5% to about30% (w/v); a metal ion source at a concentration of about 0.1 mM toabout 5 mM; and piperazine at a concentration of about 0.5 mM to about250 mM.
 2. The composition of claim 1, wherein the metal ions compriseat least one of copper ions, cobalt ions, iron ions and manganese ions.3. The composition of claim 2, wherein the metal ions comprise copperions.
 4. The composition of claim 3, wherein the copper ion source iscupric sulfate or cupric acetate.
 5. The composition of claim 4, whereinthe copper ion source is cupric sulfate.
 6. The composition of claim 1,wherein: the hydrogen peroxide is at a concentration of about 1% to 15%(w/v); the metal ion source is at a concentration of about 0.5 mM toabout 2.5 mM; and the piperazine is at a concentration of about 1 mM toabout 200 mM.
 7. The composition of claim 6, wherein the metal ionscomprise at least one of copper ions, cobalt ions, iron ions andmanganese ions.
 8. The composition of claim 7, wherein the metal ionscomprise copper ions.
 9. The composition of claim 8, wherein the copperion source is cupric sulfate or cupric acetate.
 10. The composition ofclaim 9, wherein the copper ion source is cupric sulfate.
 11. Thecomposition of claim 1, wherein: the hydrogen peroxide is at aconcentration of about 1% to about 6% (w/v); the metal ion source is ata concentration of about 1 mM to about 2.5 mM; and the piperazine is ata concentration of about 10 mM to about 100 mM.
 12. The composition ofclaim 11, wherein the metal ions comprise at least one of copper ions,cobalt ions, iron ions and manganese ions.
 13. The composition of claim12, wherein the metal ions comprise copper ions.
 14. The composition ofclaim 13, wherein the copper ion source is cupric sulfate or cupricacetate.
 15. The composition of claim 14, wherein the copper ion sourceis cupric sulfate.
 16. The composition of claim 1, wherein thecomposition comprises about 3% (w/v) hydrogen peroxide, about 2 mMcupric sulfate, and about 50 mM piperazine.
 17. The composition of claim16, wherein the pH is about 5.5.